Antibody-SN-38 immunoconjugates with a CL2A linker

ABSTRACT

The present invention concerns improved methods and compositions for preparing SN-38 conjugates of proteins or peptides, preferably immunoconjugates of antibodies or antigen-binding antibody fragments. More preferably, the SN-38 is attached to the antibody or antibody fragment using a CL2A linker, with 1-12, more preferably 6-8, alternatively 1-5 SN-38 moieties per antibody or antibody fragment. Most preferably, the immunoconjugate is prepared in large scale batches, with various modifications to the reaction scheme disclosed herein to optimize yield and recovery in large scale. Other embodiments concern optimized dosages and/or schedules of administration of immunoconjugate to maximize efficacy for disease treatment and minimize side effects of administration.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/161,806, filed May 23, 2016, which was a divisional of U.S.patent application Ser. No. 14/789,375 (now issued U.S. Pat. No.9,345,489), filed Jul. 1, 2015, which was a divisional of U.S. patentapplication Ser. No. 14/255,508 (now issued U.S. Pat. No. 9,107,960),filed Apr. 17, 2014, which was a continuation-in-part of U.S. patentapplication Ser. No. 13/948,732 (now issued U.S. Pat. No. 9,028,833),filed Jul. 23, 2013, which claimed the benefit under 35 U.S.C. 119(e) ofU.S. Provisional Patent Applications 61/736,684, filed Dec. 13, 2012,and 61/749,548, filed Jan. 7, 2013.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 27, 2016, isnamed IMM340US18_SL.txt and is 60,784 bytes in size.

FIELD OF THE INVENTION

The present invention relates to improved synthetic protocols forproducing therapeutic immunoconjugates that can target cancer cells,infectious disease organisms and/or cells associated with autoimmunedisease, which conjugates contain an antibody moiety and a drug moietyselected from the camptothecin group of drugs. The antibody and drugmoieties are linked via an intracellularly cleavable linkage thatincreases therapeutic efficacy. Most preferably, the camptothecin isSN-38 and the linker joining the antibody moiety and the drug moiety isCL2A, as described below. In particular embodiments, theimmunoconjugates may be administered at specific dosages and/orschedules of administration that provide for optimal efficacy andminimal toxicity. The optimized dosages and schedules of administrationof SN-38-conjugated antibodies for human therapeutic use disclosedherein show unexpected superior efficacy that could not have beenpredicted from animal model studies, allowing effective treatment ofcancers that are resistant to standard anti-cancer therapies, includingthe parental compound, irinotecan (CPT-11). The novel syntheticprotocols disclosed herein provide improved efficiency of synthesis ofCL2A-SN-38 conjugates, with substantially lower levels of contaminatingby-products.

BACKGROUND OF THE INVENTION

For many years it has been an aim of scientists in the field ofspecifically targeted drug therapy to use monoclonal antibodies (MAbs)for the specific delivery of toxic agents to human cancers. Conjugatesof tumor-associated MAbs and suitable toxic agents have been developed,but have had mixed success in the therapy of cancer, and virtually noapplication in other diseases, such as infectious and autoimmunediseases. The toxic agent is most commonly a chemotherapeutic drug,although particle-emitting radionuclides, or bacterial or plant toxinshave also been conjugated to MAbs, especially for the therapy of cancer(Sharkey and Goldenberg, CA Cancer J Clin. 2006 July-August;56(4):226-243) and, more recently, with radioimmunoconjugates for thepreclinical therapy of certain infectious diseases (Dadachova andCasadevall, Q J Nucl Med Mol Imaging 2006; 50(3):193-204).

The advantages of using MAb-chemotherapeutic drug conjugates are that(a) the chemotherapeutic drug itself is structurally well defined; (b)the chemotherapeutic drug is linked to the MAb protein using very welldefined conjugation chemistries, often at specific sites remote from theMAbs antigen binding regions; (c) MAb-chemotherapeutic drug conjugatescan be made more reproducibly than chemical conjugates involving MAbsand bacterial or plant toxins, and as such are more amenable tocommercial development and regulatory approval; and (d) theMAb-chemotherapeutic drug conjugates are orders of magnitude less toxicsystemically than radionuclide MAb conjugates.

Early work on protein-drug conjugates indicated that a drug preferablyis released in its original form, once it has been internalized into atarget cell, for the protein-drug conjugate to be a useful therapeutic.Trouet et al. (Proc. Natl. Acad. Sci. USA 79:626-629 (1982)) showed theadvantage of using specific peptide linkers, between the drug and theantibody moiety, which are cleaved lysosomally to liberate the intactdrug. Notably, MAb-chemotherapeutic drug conjugates prepared using mildacid-cleavable linkers, such as those containing a hydrazone, weredeveloped, based on the observation that the pH inside tumors was oftenlower than normal physiological pH (Willner et al., U.S. Pat. No.5,708,146; Trail et al. (Science 261:212-215 (1993)). The first approvedMAb-drug conjugate, gemtuzumab ozogamicin, incorporated an acid-labilehydrazone bond between an anti-CD33 antibody, humanized P67.6, and apotent calicheamicin derivative. Sievers et al., J Clin Oncol.19:3244-3254 (2001); Hamann et al., Bioconjugate Chem. 13: 47-58 (2002).In some cases, the MAb-chemotherapeutic drug conjugates were made withreductively labile hindered disulfide bonds between the chemotherapeuticdrugs and the MAb (Liu et al., Proc Natl Acad Sci USA 93: 8618-8623(1996)).

Yet another cleavable linker involves cathepsin B-labile dipeptidespacers, such as Phe-Lys or Val-Cit, similar to the lysosomally labilepeptide spacers of Trouet et al. containing from one to four aminoacids, which additionally incorporated a collapsible spacer between thedrug and the dipeptide (Dubowchik, et al., Bioconjugate Chem. 13:855-869(2002); Firestone et al., U.S. Pat. No. 6,214,345 B1; Doronina et al.,Nat Biotechnol. 21: 778-784 (2003)). The latter approaches were alsoutilized in the preparation of an immunoconjugate of camptothecin(Walker et al., Bioorg Med Chem Lett. 12:217-219 (2002)). Anothercleavable moiety that has been explored is an ester linkage incorporatedinto the linker between the antibody and the chemotherapeutic drug.Gillimard and Saragovi have found that when an ester of paclitaxel wasconjugated to anti-rat p75 MAb, MC192, or anti-human TrkA MAb, 5C3, theconjugate was found to exhibit target-specific toxicity. Gillimard andSaragovi, Cancer Res. 61:694-699 (2001).

Current notions of antibody-drug conjugate design emphasize the use ofultratoxic drugs attached to antibodies using stable bonds that arecleaved only intracellularly. This approach has been used to designconjugates of ultratoxic drugs, such as calicheamicin,monomethylauristatin-E (MMAE), and maytansinoids. Although very stablebonding to MAbs results in stability in circulation, the conjugates arealso processed in liver, spleen, and kidney, thereby releasing the toxicdrugs in those organs and potentially reducing the therapeutic window indisease treatment applications. While recent regulatory approval ofADCETRIS® (brentuximab vedotin) for Hodgkin's lymphoma and of KADCYLA®(ado-trastuzumab emtansine) for refractory breast cancer areencouraging, the lack of therapeutic efficacy in the maximumadministrable dosage of calicheamicin conjugate in non-Hodgkin lymphoma,and its subsequent discontinuation, as well as the market withdrawal ofgemtuzumab ozogamicin for AML point to the limitations of usingultratoxics in ADCs.

The conjugates of the instant invention possess greater efficacy thanunconjugated or “naked” antibodies or antibody fragments, although suchunconjugated antibody moieties have been of use in specific situations.In cancer, for example, naked antibodies have come to play a role in thetreatment of lymphomas (alemtuzumab and rituximab), colorectal and othercancers (cetuximab and bevacizumab), breast cancer (trastuzumab), aswell as a large number now in clinical development (e.g., epratuzumab,veltuzumab, milatuzumab). In most of these cases, clinical use hasinvolved combining these naked, or unconjugated, antibodies with othertherapies, such as chemotherapy or radiation therapy.

Use of CL2A linkers to attach therapeutic drugs, such as SN-38, toantibody moieties has been disclosed (e.g. U.S. Pat. Nos. 7,999,083 and8,080,250, the Examples sections incorporated herein by reference).However, a need exists for more efficient methods of preparing and usingCL2A and MAb-CL2A-SN-38 conjugates, including optimized dosage schedulesthat result in maximal efficacy and minimal toxicity, as well asefficient large scale production of CL2A-SN-38 and antibody-CL2A-SN-38conjugates.

SUMMARY OF THE INVENTION

The present invention resolves an unfulfilled need in the art byproviding improved methods and compositions for preparing andadministering camptothecin-antibody immunoconjugates. Preferably, thecamptothecin is SN-38. The disclosed methods and compositions are of usefor the treatment of a variety of diseases and conditions which arerefractory or less responsive to other forms of therapy, and can includediseases against which suitable antibodies or antigen-binding antibodyfragments for selective targeting can be developed, or are available orknown. Preferred diseases or conditions that may be treated with thesubject immunoconjugates include, for example, cancer, autoimmunedisease, immune dysfunction disease or diseases caused by infectiousorganisms.

Various embodiments of the invention relate to novel synthetic protocolsfor production of CL2A-SN-38 and mAb-CL2A-SN-38 conjugates, asexemplified in FIG. 1. Unexpectedly, the novel synthetic protocols showimproved efficiency and yield of production, with substantiallydecreased levels of contaminating by-products. In specific embodiments,the novel synthetic protocols may include use of particularintermediates, precursors, reaction steps and/or reaction conditionsfound to be advantageous in the practice of the present invention.Although the scheme shown in FIG. 1 and claim 1 is disclosed as aparticularly preferred embodiment of the synthetic protocol, the personof ordinary skill will realize that alternative schemes may be utilizedwithin the scope of the invention.

Specific steps or conditions that have been found to be unexpectedlyadvantageous in the practice of the claimed invention may include one ormore of the following. (i) In the conversion of silyl-protectedintermediate 6 (FIG. 1) to desilylated intermediate 7 (FIG. 1),tetrabutylammonium fluoride was used in a 1.1-fold molar excess, insteadof a 1.4-fold molar excess as disclosed previously (e.g., U.S. Pat. No.9,107,960). This level of the reagent was equally efficient indesilylation, while reducing the impurity profile, thereby enhancing thepurity of intermediate 7 and leading to improved yield. (ii) In theaqueous work-ups in the preparation of both intermediates 6 and 7 (FIG.1), as previously disclosed (U.S. Pat. No. 9,107,960) the work-upinvolved washing organic extracts four times with 0.05 M sodium acetatebuffer, pH 5.3 for intermediate 6 and four times with 0.25 M citratebuffer, pH 6 for intermediate 7, with water washes subsequently in eachcase. In an improved process, the buffer washes were reduced to 3 timesfor intermediate 6 and 2 times for intermediate 7, and the water washeswere removed in both cases. These modifications considerably reduced theformation of aqueous-organic emulsion, and improved product recovery inthe organic extract. (iii) The chromatographic purification ofintermediate 7 (FIG. 1) disclosed in U.S. Pat. No. 9,107,960 involvedthe use of dichloromethane-methanol mixtures for elution. In an improvedprocess, the elution was modified to use a combination ofdichloromethane-ethyl acetate-methanol mixtures with varying methanolconcentration, followed by varying concentration ofdichloromethane-methanol mixtures. This resulted in a better and afaster separation of the intermediate, and improved the recovery. Usingthese improved procedures, the overall yield in the 3-step sequence fromintermediate 4 to intermediate 7 was improved from a range of 29-40% to64% by incorporating these process changes.

The synthetic protocol for CL2A-SN-38 may further utilize one or more ofthe following improvements. (iv) In a previously disclosed syntheticprotocol (U.S. Pat. No. 9,107,960), a copper (+1)-catalyzed reactionbetween intermediates 7 and 8 (FIG. 1) to produce intermediate 9(FIG. 1) was performed using a pre-formed complex of cuprous bromide andtriphenylphosphine in dichloromethane with a tertiary amine, such asdiisopropylethyl amine, added. It was subsequently discovered that inpresence of triphenylphosphine alone, the product quality of theintermediate 7 deteriorated over time, indicating a deleterious effectof this reagent, which required its quick removal once thecopper-catalyzed reaction was completed. This has the potential toreduce the yield of purified intermediate 9 (FIG. 1). As disclosed inthe Examples below, in the present protocol the reaction was performedusing a bi-phasic mixture containing copper sulfate and ascorbic acid inwater and intermediates 7 and 8, as well as 2,6-collidine, indichloromethane. By stirring the biphasic solution overnight, typically18 h, the conversion was complete, and the crude product was separatedfrom copper sulfate and ascorbate by a simple wash with aqueous EDTA.This was followed by chromatographic purification, leading to improvedproduct yield. The modified procedure avoids use of triphenylphosphine.(v) The stability profile of intermediate 9 (FIG. 1) in solution and asisolated material without solvent showed that the product maintainedpurity of >97% either stored as solid product at −20° C. for 4 days oras a solution in dichloromethane at room temperature for 1 day. In thelatter case, storage at room temperature for 4 days reduced the purityto 89.4%, while the solution stored at −20° C. showed a purity of only92%. As previously disclosed in U.S. Pat. No. 9,107,960, the material indichloromethane, procured after chromatography and EDTA wash, was usedas such for the deprotection reaction mediated by dichloroacetic acid(DCA) and anisole. In that situation, depending on the length of storageof the intermediate 9 in solution form, the quality of the finalmaterial (product 10, which is CL2A-SN-38) varied. In an improvedprocess, intermediate 9 (FIG. 1) after purification was concentrated toa solid that was subsequently used in the final step. This modificationremoved uncertainty about changes in product quality of intermediate 9prior to the final deprotection step. A further change in this stepinvolved adding the solution of crude product, after the reaction,dropwise over several hours, into vigorously stirred tert-butyl methylether (t-BME), which enabled the precipitation of the product in theform of filterable solid. If the addition of the reaction mixture is notgradual and if the stirring is not vigorous, the product oiled outinstead of as a solid. (vi) In the conversion of intermediate 1 tointermediate 2 (FIG. 1) and in the conversion of intermediate 2 tointermediate 3 (FIG. 1), EEDQ is used as an amide coupling reagent.However, these reactions are not so limited by the specific reagentshown. One skilled in the art will recognize that the amide coupling canbe accomplished with a number of different reagents, as reviewed in: HanS-Y and Kim Y-A. Recent development of peptide coupling reagents inorganic synthesis. Tetrahedron 2004; 60: 2447-2467.

In other embodiments, the claimed invention concerns therapeutic use ofantibody conjugates of camptothecins, such as SN-38, that have nanomolartoxicities in vitro, compared to the sub-nanomolar to picomolartoxicities of ultratoxic chemotherapeutic agents like calicheamicin,maytansinoids or MMAE. Use of drugs that are not ultratoxic allows theuse of antibody-drug linkers that do not require cell internalizationfor the release of free drugs, but rather allow some extracellularrelease of drug. With the CL2A linker described herein, 50% of theconjugated drug is released in 24 hr, thereby augmenting thebioavailability of the drug by liberating it both extracellularly andintracellularly. In addition, the use of relatively non-toxic drugsallows the administration of higher dosages of ADCs, leading to bettertherapeutic effects.

The antibody incorporated in the subject immunoconjugates can be ofvarious isotypes, preferably human IgG1, IgG2, IgG3 or IgG4, morepreferably comprising human IgG1 hinge and constant region sequences.The antibody or fragment thereof can be a chimeric, humanized, or fullyhuman antibody or antigen-binding fragment thereof, such as half-IgG4antibodies, as described by van der Neut Kolfschoten et al. (Science2007; 317:1554-1557), or single domain antibodies (e.g., nanobodies) ascommercially available (e.g., ABLYNX®, Ghent, Belgium). More preferably,the antibody or fragment thereof may be designed or selected to comprisehuman constant region sequences that belong to specific allotypes, whichmay result in reduced immunogenicity when the immunoconjugate isadministered to a human subject. Preferred allotypes for administrationinclude a non-G1m1 allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 orG1m3,1,2. More preferably, the allotype is selected from the groupconsisting of the nG1m1, G1m3, nG1m1,2 and Km3 allotypes.

Antibodies of use may bind to any disease-associated antigen known inthe art. Where the disease state is cancer, for example, many antigensexpressed by or otherwise associated with tumor cells are known in theart, including but not limited to, carbonic anhydrase IX,alpha-fetoprotein (AFP), α-actinin-4, A3, antigen specific for A33antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1,CASP-8/m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14,CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b,CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59,CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126,CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4,CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp), CEACAM5,CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M,Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor,G250 antigen, GAGE, gp100, GRO -β, HLA-DR, HM1.24, human chorionicgonadotropin (HCG) and its subunits, HER2/neu, histone H2B, histone H3,histone H4, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2,Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R,IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23,IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen,KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF),MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A,MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2,MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD-1, PD-L1, PD-1receptor, placental growth factor, p53, PLAGL2, prostatic acidphosphatase, PSA, PRAIVIE, PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5,RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin,TRAIL receptors, TNF-α, Tn antigen, Thomson-Friedenreich antigens, tumornecrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen,complement factors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2,bcl-6, Kras, an oncogene marker and an oncogene product (see, e.g.,Sensi et al., Clin Cancer Res 2006, 12:5023-32; Parmiani et al., JImmunol 2007, 178:1975-79; Novellino et al. Cancer Immunol Immunother2005, 54:187-207). Preferably, the antibody binds to AFP, CEACAM5,CEACAM6, CSAp, EGP-1 (TROP-2), AFP, MUC5ac, CD74, CD19, CD20, CD22 orHLA-DR.

Exemplary antibodies that may be utilized include, but are not limitedto, hR1 (anti-IGF-1R, U.S. patent application Ser. No. 13/688,812, filedNov. 29, 2012), hPAM4 (anti-MUC5ac, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,151,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.5,789,554), hRFB4 (anti-CD22, U.S. Pat. No. 9,139,649), hMu-9(anti-CSAp, U.S. Pat. No. 7,387,773), hL243 (anti-HLA-DR, U.S. Pat. No.7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat. No. 6,676,924), hMN-15(anti-CEACAM6, U.S. Pat. No. 8,287,865), hRS7 (anti-EGP-1, U.S. Pat. No.7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat. No. 7,541,440), Ab124 andAb125 (anti-CXCR4, U.S. Pat. No. 7,138,496), the Examples section ofeach cited patent or application incorporated herein by reference. Morepreferably, the antibody is IMMU-31 (anti-AFP), hRS7 (anti-TROP-2),hMN-14 (anti-CEACAM5), hMN-3 (anti-CEACAM6), hMN-15 (anti-CEACAM6), hLL1(anti-CD74), hLL2 (anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19(anti-CD19) or hA20 (anti-CD20). As used herein, the terms epratuzumaband hLL2 are interchangeable, as are the terms veltuzumab and hA20,hL243g4P, hL243gamma4P and IMMU-114. In specific preferred embodiments,the immunoconjugate may be an hMN-14-SN-38, hMN-3-SN-38, hMN-15-SN-38,hIMMU-31-SN-38, hRS7-SN-38, hR1-SN-38, hA20-SN-38, hPAM4-SN-38,hL243-SN-38, hLL1-SN-38, hRFB4-SN-38, hMu-9-SN-38 or hLL2-SN-38conjugate with a CL2A linker.

Alternative antibodies of use include, but are not limited to, abciximab(anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab(anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab(anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1receptor), nivolumab (anti-PD-1 receptor), ipilimumab (anti-CTLA-4),abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab(anti-IL-6 receptor), benralizumab (anti-CD125), obinutuzumab (GA101,anti-CD20), CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti -PSMA, U.S. patentapplication Ser. No. 11/983,372, deposited as ATCC PTA-4405 andPTA-4406), D2/B (anti -PSMA, WO 2009/130575), tocilizumab (anti-IL-6receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (anti-CD3receptor), natalizumab (anti-α4 integrin), omalizumab (anti-IgE);anti-TNF-α antibodies such as CDP571 (Ofei et al., 2011, Diabetes45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (ThermoScientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.),certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels,Belgium), adalimumab (Abbott, Abbott Park, Ill.), Benlysta (Human GenomeSciences); antibodies for therapy of Alzheimer's disease such as Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,solanezumab and infliximab; anti-fibrin antibodies like 59D8, T2G1s,MH1; anti-CD38 antibodies such as MOR03087 (MorphoSys AG), MOR202(Celgene), HuMax-CD38 (Genmab) or daratumumab (Johnson & Johnson);(anti-HIV antibodies such as P4/D10 (U.S. Pat. No. 8,333,971), Ab 75, Ab76, Ab 77 (Paulik et al., 1999, Biochem Pharmacol 58:1781-90), as wellas the anti-HIV antibodies described and sold by Polymun (Vienna,Austria), also described in U.S. Pat. No. 5,831,034, U.S. Pat. No.5,911,989, and Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos etal., Antimicrob. Agents Chemother. 2006; 50(5):1773-9, all incorporatedherein by reference; and antibodies against pathogens such as CR6261(anti-influenza), exbivirumab (anti-hepatitis B), felvizumab(anti-respiratory syncytial virus), foravirumab (anti-rabies virus),motavizumab (anti-respiratory syncytial virus), palivizumab(anti-respiratory syncytial virus), panobacumab (anti-Pseudomonas),rafivirumab (anti-rabies virus), regavirumab (anti-cytomegalovirus),sevirumab (anti-cytomegalovirus), tivirumab (anti-hepatitis B), andurtoxazumab (anti-E. coli).

Preferably, the antibody moiety links to at least one drug moiety, morepreferably 1 to about 5 drug moieties, alternatively about 7 to 12 drugmoieties. In various embodiments, the antibody moiety may be attached to4 to 6 drug moieties, 6 to 8 drug moieties or 7 to 8 drug moieties. Thenumber of drug moieties per antibody moiety may be 1, 2, 3, 4, 5, 6, 7,8 or more.

Various embodiments may concern use of the subject methods andcompositions to treat a cancer, including but not limited tonon-Hodgkin's lymphomas, B-cell acute and chronic lymphoid leukemias,Burkitt lymphoma, Hodgkin's lymphoma, acute large B-cell lymphoma, hairycell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia, T-cell lymphomas andleukemias, multiple myeloma, Waldenstrom's macroglobulinemia,carcinomas, melanomas, sarcomas, gliomas, bone, and skin cancers. Thecarcinomas may include carcinomas of the oral cavity, esophagus,gastrointestinal tract, pulmonary tract, lung, stomach, colon, breast,ovary, prostate, uterus, endometrium, cervix, urinary bladder, pancreas,bone, brain, connective tissue, liver, gall bladder, urinary bladder,kidney, skin, central nervous system and testes.

Exemplary autoimmune or immune dysfunction diseases that may potentiallybe treated with the subject immunoconjugates include acute immunethrombocytopenia, chronic immune thrombocytopenia, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, pemphigus vulgaris, diabetes mellitus (e.g., juvenilediabetes), Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosum, Takayasu's arteritis, ANCA-associated vasculitides,Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis obliterans, Sjögren's syndrome, primary biliarycirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronicactive hepatitis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis, fibrosing alveolitis, graft-versus-hostdisease (GVHD), organ transplant rejection, sepsis, septicemia andinflammation.

In addition, the subject methods and compositions may be used to treatan infectious disease, for example diseases involving infection bypathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi,viruses, parasites, or other microbial agents. Examples include humanimmunodeficiency virus (HIV) causing AIDS, Mycobacterium oftuberculosis, Streptococcus agalactiae, methicillin-resistantStaphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes,Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis,Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum,Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes,West Nile virus, Pseudomonas aeruginosa, Mycobacterium leprae, Brucellaabortus, rabies virus, influenza virus, cytomegalovirus, herpes simplexvirus I, herpes simplex virus II, human serum parvo-like virus,respiratory syncytial virus, varicella-zoster virus, hepatitis B virus,hepatitis C virus, measles virus, adenovirus, human T-cell leukemiaviruses, Epstein-Barr virus, murine leukemia virus, mumps virus,vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitisvirus, wart virus, blue tongue virus, Sendai virus, feline leukemiavirus, reo virus, polio virus, simian virus 40, mouse mammary tumorvirus, dengue virus, rubella virus, Plasmodium falciparum, Plasmodiumvivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi,Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni,Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocercavolvulus, Leishmania tropica, Trichinella spiralis, Theileria parva,Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcusgranulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis,M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M.pneumoniae. A review listing antibodies against infectious organisms(antitoxin and antiviral antibodies), as well as other targets, iscontained in Casadevall, Clin Immunol 1999; 93(1):5-15, incorporatedherein by reference.

In certain embodiments involving treatment of cancer, the drugconjugates may be used in combination with another therapeutic modality,such as surgery, radiation therapy, chemotherapy, immunotherapy withnaked antibodies, radioimmunotherapy, immunomodulators, or vaccines.Similar combination therapies may be used in the treatment of otherdiseases amenable to antibody moieties, such as autoimmune diseases. Forexample, camptothecin conjugates can be combined with TNF inhibitors,B-cell antibodies, interferons, interleukins, and other effective agentsfor the treatment of autoimmune diseases, such as rheumatoid arthritis,systemic lupus erythematosis, Sjögren's syndrome, multiple sclerosis,vasculitis, as well as type-I diabetes (juvenile diabetes). Thesecombination therapies can allow lower doses of each therapeutic to begiven in such combinations, thus reducing certain severe side effects,and potentially reducing the courses of therapy required.

In infectious diseases, the drug immunoconjugates can be combined withother therapeutic drugs, immunomodulators, naked MAbs, or vaccines(e.g., MAbs against hepatitis, HIV, or papilloma viruses, or vaccinesbased on immunogens of these viruses, or kinase inhibitors, such as inhepatitis B). Antibodies and antigen-based vaccines against these andother viral pathogens are known in the art and, in some cases, alreadyin commercial use. The development of anti-infective monoclonalantibodies has been reviewed recently by Reichert and Dewitz (Nat RevDrug Discovery 2006; 5:191-195), incorporated herein by reference, whichsummarizes the priority pathogens against which naked antibody therapyhas been pursued, resulting in only 2 pathogens against which antibodiesare either in Phase III clinical trials or are being marketed(respiratory syncytial virus and methicillin-resistant Staphylococcusaureus), with 25 others in clinical studies and 20 discontinued duringclinical study. For combination therapy, the use of radioimmunotherapyfor the treatment of infectious organisms is disclosed, for example, inU.S. Pat. Nos. 4,925,648; 5,332,567; 5,439,665; 5,601,825; 5,609,846;5,612,016; 6,120,768; 6,319,500; 6,458,933; 6,548,275; and in U.S.Patent Application Publication Nos. 20020136690 and 20030103982, theExamples section of each of which is incorporated herein by reference.

Preferred optimal dosing of immunoconjugates may include a dosage ofbetween 3 mg/kg and 18 mg/kg, preferably given either weekly, twiceweekly, every other week or every third week. The optimal dosingschedule may include treatment cycles of two consecutive weeks oftherapy followed by one, two, three or four weeks of rest, oralternating weeks of therapy and rest, or one week of therapy followedby two, three or four weeks of rest, or three weeks of therapy followedby one, two, three or four weeks of rest, or four weeks of therapyfollowed by one, two, three or four weeks of rest, or five weeks oftherapy followed by one, two, three, four or five weeks of rest, oradministration once every two weeks, once every three weeks or once amonth. Treatment may be extended for any number of cycles, preferably atleast 2, at least 4, at least 6, at least 8, at least 10, at least 12,at least 14, or at least 16 cycles. Exemplary dosages of use may include1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg,9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22 mg/kg and 24 mg/kg.Preferred dosages are 4, 6, 8, 9, 10, 12, 14, 16 or 18 mg/kg. Mostpreferred dosages are 8 or 10 mg/kg. The person of ordinary skill willrealize that a variety of factors, such as age, general health, specificorgan function or weight, as well as effects of prior therapy onspecific organ systems (e.g., bone marrow) may be considered inselecting an optimal dosage of immunoconjugate, and that the dosageand/or frequency of administration may be increased or decreased duringthe course of therapy. The dosage may be repeated as needed, withevidence of tumor shrinkage observed after as few as 4 to 8 doses. Theoptimized dosages and schedules of administration disclosed herein showunexpected superior efficacy and reduced toxicity in human subjects,which could not have been predicted from animal model studies.Surprisingly, the superior efficacy allows treatment of tumors that werepreviously found to be resistant to one or more standard anti-cancertherapies, including the parental compound, CPT-11, from which SN-38 isderived in vivo.

A surprising result with the instant claimed compositions and methods isthe unexpected tolerability of high doses of antibody-drug conjugate,even with repeated infusions, with only relatively low-grade toxicitiesof nausea, vomiting and diarrhea observed, as well as skin rash, ormanageable neutropenia. A further surprising result is the lack ofaccumulation of the antibody-drug conjugate, unlike other products thathave conjugated SN-38 to albumin, PEG or other carriers. The lack ofaccumulation is associated with improved tolerability and lack ofserious toxicity even after repeated or increased dosing. Thesesurprising results allow optimization of dosage and delivery schedule,with unexpectedly high efficacies and low toxicities. The claimedmethods provide for shrinkage of tumors, in individuals with previouslyresistant cancers, of 15% or more, preferably 20% or more, preferably30% or more, more preferably 40% or more in size (as measured by longestdiameter). The person of ordinary skill will realize that tumor size maybe measured by a variety of different techniques, such as total tumorvolume, maximal tumor size in any dimension or a combination of sizemeasurements in several dimensions. This may be with standardradiological procedures, such as computed tomography, ultrasonography,and/or positron-emission tomography. The means of measuring size is lessimportant than observing a trend of decreasing tumor size withimmunoconjugate treatment, preferably resulting in elimination of thetumor.

While the immunoconjugate may be administered as a periodic bolusinjection, in alternative embodiments the immunoconjugate may beadministered by continuous infusion of antibody-drug conjugates. Inorder to increase the Cmax and extend the PK of the immunoconjugate inthe blood, a continuous infusion may be administered for example byindwelling catheter. Such devices are known in the art, such asHICKMAN®, BROVIAC® or PORT-A-CATH® catheters (see, e.g., Skolnik et al.,Ther Drug Monit 32:741-48, 2010) and any such known indwelling cathetermay be used. A variety of continuous infusion pumps are also known inthe art and any such known infusion pump may be used. The dosage rangefor continuous infusion may be between 0.1 and 2.0 mg/kg per day. Morepreferably, these immunoconjugates can be administered by intravenousinfusions over relatively short periods of 2 to 5 hours, more preferably2-3 hours.

In particularly preferred embodiments, the immunoconjugates and dosingschedules may be efficacious in patients resistant to standardtherapies. For example, a mAb-CL2A-SN-38 immunoconjugate may beadministered to a patient who has not responded to prior therapy withirinotecan, the parent agent of SN-38. Surprisingly, theirinotecan-resistant patient may show a partial response tomAb-CL2A-SN-38. The ability of the immunoconjugate to specificallytarget the tumor tissue may overcome tumor resistance by improvedtargeting and enhanced delivery of the therapeutic agent. Combinationsof different SN-38 immunoconjugates, or SN-38-antibody conjugates incombination with an antibody conjugated to a radionuclide, toxin orother drug, may provide even more improved efficacy and/or reducedtoxicity. A specific preferred subject may be a metastatic colon cancerpatient, a triple-negative breast cancer patient, a HER+, ER+,progesterone+ breast cancer patient, a metastatic non-small-cell lungcancer (NSCLC) patient, a metastatic pancreatic cancer patient, ametastatic renal cell carcinoma patient, a metastatic gastric cancerpatient, a metastatic prostate cancer patient, or a metastaticsmall-cell lung cancer patient. In different embodiments, the therapymay be suitable to treat front-line, second-line, third-line or otherpatients.

Certain embodiments relate to improved methods for preparing CL2A-SN38conjugates and antibody-CL2A-SN-38 immunoconjugates in large scale, withimproved yield and/or efficiency. A particularly preferred embodiment isillustrated in the synthetic scheme shown in FIG. 1. Preferably, themaleimide group at the end of the CL2A linker reacts with sulfhydrylside chains on reduced cysteine residues on an antibody moiety or othertargeting peptide or protein, although other methods of attaching theCL2A moiety to the antibody moiety are known and may be used. In afurther preferred embodiment, the immunoconjugates prepared from theCL2A-SN-38 moiety are purified by tangential flow filtration, therebyavoiding cumbersome chromatography on size-exclusion andhydrophobic-interaction columns, and enabling high protein recoveriesafter purification.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Novel scheme for improved large-scale production of CL2A-SN-38.

FIG. 2. In vivo therapy of athymic nude mice, bearing Capan 1 humanpancreatic carcinoma, with MAb-CL2A-SN-38 conjugates.

FIG. 3. In vivo therapy of athymic nude mice, bearing BxPC3 humanpancreatic carcinoma, with MAb-CL2A-SN-38 conjugates.

FIG. 4. In vivo therapy of athymic nude mice, bearing LS174T human coloncarcinoma, with hMN-14-CL2A-SN-38 conjugate.

FIG. 5A. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humannon-small cell lung tumor xenografts. Mice bearing Calu-3 tumors (N=5-7)were injected with hRS7-CL2-SN-38 every 4 days for a total of 4injections (q4dx4). All the ADCs and controls were administered in theamounts indicated (expressed as amount of SN-38 per dose; longarrows=conjugate injections, short arrows=irinotecan injections).

FIG. 5B. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humancolorectal tumor xenografts. COLO 205 tumor-bearing mice (N=5) wereinjected 8 times (q4dx8) with the ADC or every 2 days for a total of 5injections (q2dx5) with the MTD of irinotecan. All the ADCs and controlswere administered in the amounts indicated (expressed as amount of SN-38per dose; long arrows=conjugate injections, short arrows=irinotecaninjections).

FIG. 5C. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humanpancreatic cancer xenografts. Capan-1 (N=10) tumor-bearing mice (N=10)were treated twice weekly for 4 weeks with the agents indicated. All theADCs and controls were administered in the amounts indicated (expressedas amount of SN-38 per dose; long arrows=conjugate injections, shortarrows=irinotecan injections).

FIG. 5D. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humanpancreatic cancer xenografts. BxPC-3 tumor-bearing mice (N=10) weretreated twice weekly for 4 weeks with the agents indicated. All the ADCsand controls were administered in the amounts indicated (expressed asamount of SN-38 per dose; long arrows=conjugate injections, shortarrows=irinotecan injections).

FIG. 5E. Therapeutic efficacy of hRS7-SN-38 ADC in mice bearing humansquamous cell lung carcinoma xenografts. In addition to ADC given twiceweekly for 4 week, SK-MES-1 tumor-bearing (N=8) mice received the MTD ofCPT-11 (q2dx5). All the ADCs and controls were administered in theamounts indicated (expressed as amount of SN-38 per dose; longarrows=conjugate injections, short arrows=irinotecan injections).

FIG. 6. Comparative efficacy of epratuzumab (Emab)-SN-38 and veltuzumab(Vmab)-SN-38 conjugates in the subcutaneous Ramos model. Nude mice (N=10per group) with tumors averaging approximately 0.35 cm³ (0.20-0.55 cm³)were administered 0.25 or 0.5 mg of each conjugate twice weekly for 4weeks.

FIG. 7A. Specificity of Emab anti-CD22-SN-38 conjugate (solid line)versus an irrelevant labetuzumab (Lmab)-SN-38 conjugate (dashed line) innude mice bearing subcutaneous Ramos tumors. Animals were given twiceweekly doses of 75 μg of each conjugate per dose (54.5 μg/kg of SN-38,based on average weight of 22 g) intraperitoneally for 4 weeks. Survivalbased on time-to-progression (TTP) to 3.0 cm³, with tumors starting atan average size of 0.4 cm³. P values comparing median survival (shown)for Emab-SN-38 to Lmab-SN-38 conjugate are shown in each panel.

FIG. 7B. Specificity of Emab anti-CD22-SN-38 conjugate (solid line)versus an irrelevant labetuzumab (Lmab)-SN-38 conjugate (dashed line) innude mice bearing subcutaneous Ramos tumors. Animals were given twiceweekly doses of 125 μg of each conjugate per dose (91 μg/kg of SN-38,based on average weight of 22 g) intraperitoneally for 4 weeks. Survivalbased on time-to-progression (TTP) to 3.0 cm³, with tumors starting atan average size of 0.4 cm³. P values comparing median survival (shown)for Emab-SN-38 to Lmab-SN-38 conjugate are shown in each panel.

FIG. 7C. Specificity of Emab anti-CD22-SN-38 conjugate (solid line)versus an irrelevant labetuzumab (Lmab)-SN-38 conjugate (dashed line) innude mice bearing subcutaneous Ramos tumors. Animals were given twiceweekly doses of 250 μg of each conjugate per dose (182 μg/kg of SN-38,based on average weight of 22 g) intraperitoneally for 4 weeks. Survivalbased on time-to-progression (TTP) to 3.0 cm³, with tumors starting atan average size of 0.4 cm³. P values comparing median survival (shown)for Emab-SN-38 to Lmab-SN-38 conjugate are shown in each panel. Survivalcurves (solid gray) are also shown for another group of animals givenweekly intraperitoneal injections of irinotecan (6.5 μg/dose; SN-38equivalents approximately the same as the 250-μg dose of the Emab-SN-38conjugate).

FIG. 8. History of prior treatment of patient, before administeringIMMU-130 (labetuzumab-NS-38). Prior treatment included stage IV CRCcolectomy/hepatectomy (partial lobe), radiofrequency ablation therapy ofliver metasteses, wedge resection of lung metasteses, and chemotherapywith irinotecan/oxaliplatin, Folfirinox, Folfirinox+bevacizumab,bevacizumab+5-FU/leucovorin, FolFiri, Folfiri+cetuximab, and cetuximabalone. The patient received doses of 16 mg/kg of IMMU-132 by slow IVinfusion every other week for a total of 17 treatment doses.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of theclaimed subject matter. Terms that are not expressly defined herein areused in accordance with their plain and ordinary meanings.

Unless otherwise specified, a or an means “one or more.”

The term about is used herein to mean plus or minus ten percent (10%) ofa value. For example, “about 100” refers to any number between 90 and110.

An antibody, as used herein, refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an antigen-binding portion of an immunoglobulin molecule,such as an antibody fragment. An antibody or antibody fragment may beconjugated or otherwise derivatized within the scope of the claimedsubject matter. Such antibodies include but are not limited to IgG1,IgG2, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv), single domain antibodies(DABs or VHHs) and the like, including the half-molecules of IgG4 citedabove (van der Neut Kolfschoten et al. (Science 2007; 317(14September):1554-1557). A commercially available form of single domainantibody, referred to as a nanobody (ABLYNX®, Ghent, Belgium), isdiscussed in further detail below. Regardless of structure, an antibodyfragment of use binds with the same antigen that is recognized by theintact antibody. The term “antibody fragment” also includes synthetic orgenetically engineered proteins that act like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains, recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”), and minimal recognition units consisting of the aminoacid residues that mimic the hypervariable region, such as CDRs. The Fvfragments may be constructed in different ways to yield multivalentand/or multispecific binding forms. In the case of multivalent, theyhave more than one binding site against the specific epitope, whereaswith multispecific forms, more than one epitope (either of the sameantigen or against one antigen and a different antigen) is bound.

A naked antibody is generally an entire antibody that is not conjugatedto a therapeutic agent. This is so because the Fc portion of theantibody molecule provides effector or immunological functions, such ascomplement fixation and ADCC (antibody-dependent cell cytotoxicity),which set mechanisms into action that may result in cell lysis. However,the Fc portion may not be required for therapeutic function of theantibody, but rather other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,such as inhibition of heterotypic or homotypic adhesion, andinterference in signaling pathways, may come into play and interferewith disease progression. Naked antibodies include both polyclonal andmonoclonal antibodies, and fragments thereof, that include murineantibodies, as well as certain recombinant antibodies, such as chimeric,humanized or human antibodies and fragments thereof. As used herein,“naked” is synonymous with “unconjugated,” and means not linked orconjugated to a therapeutic agent.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, more preferably a murineantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a primate, cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a murine antibody, are transferred fromthe heavy and light variable chains of the murine antibody into humanheavy and light variable domains (framework regions). The constantdomains of the antibody molecule are derived from those of a humanantibody. In some cases, specific residues of the framework region ofthe humanized antibody, particularly those that are touching or close tothe CDR sequences, may be modified, for example replaced with thecorresponding residues from the original murine, rodent, subhumanprimate, or other antibody.

A human antibody is an antibody obtained, for example, from transgenicmice that have been “engineered” to produce human antibodies in responseto antigenic challenge. In this technique, elements of the human heavyand light chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for various antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, the Examples section of eachof which is incorporated herein by reference.

Infectious Diseases as used herein are diseases involving infection bypathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi,viruses, parasites, or other microbial agents. Examples include humanimmunodeficiency virus (HIV) causing AIDS, Mycobacterium oftuberculosis, Streptococcus agalactiae, methicillin-resistantStaphylococcus aureus, Legionella pneumophilia, Streptococcus pyogenes,Escherichia coli, Neisseria gonorrhosae, Neisseria meningitidis,Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum,Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes,West Nile virus, Pseudomonas aeruginosa, Mycobacterium leprae, Brucellaabortus, rabies virus, influenza virus, cytomegalovirus, herpes simplexvirus I, herpes simplex virus II, human serum parvo-like virus,respiratory syncytial virus, varicella-zoster virus, hepatitis B virus,hepatitis C virus, measles virus, adenovirus, human T-cell leukemiaviruses, Epstein-Barr virus, murine leukemia virus, mumps virus,vesicular stomatitis virus, sindbis virus, lymphocytic choriomeningitisvirus, wart virus, blue tongue virus, Sendai virus, feline leukemiavirus, reo virus, polio virus, simian virus 40, mouse mammary tumorvirus, dengue virus, rubella virus, Plasmodium falciparum, Plasmodiumvivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi,Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni,Schistosoma japanicum, Babesia bovis, Elmeria tenella, Onchocercavolvulus, Leishmania tropica, Trichinella spiralis, Theileria parva,Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcusgranulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis,M. orate, M. arginini, Acholeplasma laidlawii, M. salivarium and M.pneumoniae. A review listing antibodies against infectious organisms(antitoxin and antiviral antibodies), as well as other targets, iscontained in Casadevall, Clin Immunol 1999; 93(1):5-15, incorporatedherein by reference.

A therapeutic agent is a molecule or atom that is administeredseparately, concurrently or sequentially with a binding moiety, e.g., anantibody or antibody fragment, and is useful in the treatment of adisease. Examples of therapeutic agents include, but are not limited to,antibodies, antibody fragments, conjugates, drugs, cytotoxic agents,proapoptotic agents, toxins, nucleases (including DNAses and RNAses),hormones, immunomodulators, chelators, boron compounds, photoactiveagents or dyes, radioisotopes or radionuclides, oligonucleotides,interference RNA, peptides, anti-angiogenic agents, chemotherapeuticagents, cyokines, chemokines, prodrugs, enzymes, binding proteins orpeptides or combinations thereof.

An immunoconjugate is an antibody, antibody fragment or other antibodymoiety conjugated to a therapeutic agent. As used herein, the terms“conjugate” and “immunoconjugate” are used interchangeably.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which one or morenatural antibodies, single-chain antibodies or antibody fragments arelinked to another moiety, such as a protein or peptide, a toxin, acytokine, a hormone, etc. In certain preferred embodiments, the fusionprotein may comprise two or more of the same or different antibodies,antibody fragments or single-chain antibodies fused together, which maybind to the same epitope, different epitopes on the same antigen, ordifferent antigens.

An immunomodulator is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use stimulates immune cells to proliferate or becomeactivated in an immune response cascade, such as macrophages, dendriticcells, B-cells, and/or T-cells. However, in some cases animmunomodulator may suppress proliferation or activation of immunecells, as in therapeutic treatment of autoimmune disease. An example ofan immunomodulator as described herein is a cytokine, which is a solublesmall protein of approximately 5-20 kDa that is released by one cellpopulation (e.g., primed T-lymphocytes) on contact with specificantigens, and which acts as an intercellular mediator between cells. Asthe skilled artisan will understand, examples of cytokines includelymphokines, monokines, interleukins, and several related signalingmolecules, such as tumor necrosis factor (TNF) and interferons.Chemokines are a subset of cytokines. Certain interleukins andinterferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation.

CPT is an abbreviation for camptothecin. As used in the presentapplication, CPT represents camptothecin itself or an analog orderivative of camptothecin. The structures of camptothecin and some ofits analogs, with the numbering indicated and the rings labeled withletters A-E, are given in formula 1 in Chart 1 below.

ABBREVIATIONS

DCA—Dicloroacetic acid

Fmoc—Fluorenylmethyloxycarbonyl chloride

EEDQ—2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline

MCC-yne—4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide

MMT—Monomethoxytrityl

PABOH—p-Aminobenzyl alcohol

PEG—Polyethylene glycol

TBAF—Tetra-n-butylammonium fluoride

TBDMS—tert-butyldimethylsilyl

Camptothecin Conjugates

Non-limiting methods and compositions for preparing immunoconjugatescomprising a camptothecin therapeutic agent attached to an antibody orantigen-binding antibody fragment are described below. In preferredembodiments, the solubility of the drug is enhanced by placing a definedpolyethyleneglycol (PEG) moiety (i.e., a PEG containing a defined numberof monomeric units) between the drug and the antibody, wherein thedefined PEG is a low molecular weight PEG, preferably containing 1-30monomeric units, more preferably containing 1-12 monomeric units, mostpreferably containing 8 monomeric units.

Preferably, a first linker connects the drug at one end and mayterminate with an acetylene or an azide group at the other end. Thisfirst linker may comprise a defined PEG moiety with an azide oracetylene group at one end and a different reactive group, such ascarboxylic acid or hydroxyl group, at the other end. Said bifunctionaldefined PEG may be attached to the amine group of an amino alcohol, andthe hydroxyl group of the latter may be attached to the hydroxyl groupon the drug in the form of a carbonate. Alternatively, the non-azide (oracetylene) moiety of said defined bifunctional PEG may be attached tothe N-terminus of an L-amino acid or a polypeptide, with the C-terminusattached to the amino group of amino alcohol, and the hydroxy group ofthe latter may be attached to the hydroxyl group of the drug in the formof carbonate or carbamate, respectively.

A second linker, comprising an antibody-coupling group and a reactivegroup complementary to the azide (or acetylene) group of the firstlinker, namely acetylene (or azide), may react with the drug-(firstlinker) conjugate via acetylene-azide cycloaddition reaction to furnisha final bifunctional drug product that is useful for conjugating todisease-targeting antibodies. The antibody-coupling group is preferablyeither a thiol or a thiol-reactive group.

Methods for selective regeneration of the 10-hydroxyl group in thepresence of the C-20 carbonate in preparations of drug-linker precursorinvolving CPT analogs such as SN-38 are provided below. Other protectinggroups for reactive hydroxyl groups in drugs such as the phenolichydroxyl in SN-38, for example t-butyldimethylsilyl ort-butyldiphenylsilyl, may also be used, and these may be deprotected bytetrabutylammonium fluoride prior to linking of the derivatized drug toan antibody-coupling moiety. The 10-hydroxyl group of CPT analogs isalternatively protected as an ester or carbonate, other than ‘BOC’, suchthat the bifunctional CPT is conjugated to an antibody without priordeprotection of this protecting group. The protecting group may bereadily deprotected under physiological pH conditions after thebioconjugate is administered.

In the acetylene-azide coupling, referred to as ‘click chemistry’, theazide part may be on L2 with the acetylene part on L3. Alternatively, L2may contain acetylene, with L3 containing azide. ‘Click chemistry’refers to a copper (+1)-catalyzed cycloaddition reaction between anacetylene moiety and an azide moiety (Kolb H C and Sharpless K B, DrugDiscov Today 2003; 8: 1128-37), although alternative forms of clickchemistry are known and may be used. The reaction may use a mixture ofcuprous bromide and triphenylphosphine to enable highly efficientcoupling in non-polar organic solvents, such as dichloromethane.However, as disclosed below, triphosphine is disadvantageous for certainpurposes and alternative methods to facilitate the click coupling stepmay be utilized. The advantage of click chemistry is that it ischemoselective, and complements other well-known conjugation chemistriessuch as the thiol-maleimide reaction. In the following discussion, wherea conjugate comprises an antibody or antibody fragment, another type ofbinding moiety, such as a targeting peptide, may be substituted.

An exemplary preferred embodiment is directed to a conjugate of a drugderivative and an antibody of the general formula 2,MAb-[L2]-[L1]-[AA] _(m-) [A′]-Drug  (2)where MAb is a disease-targeting antibody; L2 is a component of thecross-linker comprising an antibody-coupling moiety and one or more ofacetylene (or azide) groups; L1 comprises a defined PEG with azide (oracetylene) at one end, complementary to the acetylene (or azide) moietyin L2, and a reactive group such as carboxylic acid or hydroxyl group atthe other end; AA is an L-amino acid; m is an integer with values of 0,1, 2, 3, or 4 (preferably 1); and A′ is an additional spacer, selectedfrom the group of ethanolamine, 4-hydroxybenzyl alcohol, 4-aminobenzylalcohol, or substituted or unsubstituted ethylenediamine. The L aminoacids of ‘AA’ are selected from alanine, arginine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine. If the A′ group containshydroxyl, it is linked to the hydroxyl group or amino group of the drugin the form of a carbonate or carbamate, respectively.

In a preferred embodiment of formula 2, ‘A’ of the general formula 2 isA-OH, whereby A-OH is a collapsible moiety such as 4-aminobenzyl alcoholor a substituted 4-aminobenzyl alcohol substituted with a C₁-C₁₀ alkylgroup at the benzylic position, and the latter, via its amino group, isattached to an L-amino acid or a polypeptide comprising up to fourL-amino acid moieties; wherein the N-terminus is attached to across-linker terminating in the antibody moiety-binding group.

An example of a preferred embodiment is given below, wherein the A-OHembodiment of A′ of general formula (2) is derived from substituted4-aminobenzyl alcohol, and ‘AA’ is comprised of a single L-amino acidwith m=1 in the general formula (2), and the drug is exemplified withSN-38. The structure is represented below (formula 3, referred to asMAb-CLX-SN-38). Single amino acid of AA is selected from any one of thefollowing L-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. The substituent R on 4-aminobenzylalcohol moiety (A-OH embodiment of A′) is hydrogen or an alkyl groupselected from C1-C10 alkyl groups.

An embodiment of MAb-CLX-SN-38 of formula 3, wherein the single aminoacid AA is L-lysine and R═H, and the drug is exemplified by SN-38(formula 4; referred to as MAb-CL2A-SN-38).

In another preferred embodiment, the L1 component of the conjugatecontains a defined polyethyleneglycol (PEG) spacer with 1-30 repeatingmonomeric units. In a further preferred embodiment, PEG is a defined PEGwith 1-12 (more preferably 8) repeating monomeric units. Theintroduction of PEG may involve using heterobifunctionalized PEGderivatives which are available commercially. The heterobifunctional PEGmay contain an azide or acetylene group. An example of aheterobifunctional defined PEG containing 8 repeating monomeric units,with ‘NHS’ being succinimidyl, is given below in formula 5:

In a preferred embodiment, the process of producing CL2A-SN-38 is asshown in FIG. 1. In this embodiment, certain significant process changeshave been made in the preparation of CL2A-SN-38 used to make theimmunoconjugate MAb-CL2A-SN-38. These changes to the proceduresdisclosed, for example, in U.S. Pat. No. 7,999,083, not obvious to oneof ordinary skill in the art, comprise the following changes.

-   -   1) Although hexane was previously used to precipitate        Lys(MMT)-PABOH (intermediate 2 in FIG. 1), heptane was        substituted for hexane as a safer alternative. Residual        unremoved diethylamine was assayed by NMR spectroscopy. If the        presence of diethylamine was detected, the material was further        purified to remove diethylamine, which avoided the reduction in        yield caused by the presence of diethylamine in intermediate 2.    -   2) In the preparation of 10-O-TBDMS-SN-38 (intermediate 4 in        FIG. 1) from SN-38, dimethylformamide (DMF) that was previously        used as solvent for the reaction was replaced with        dichloromethane (DCM), thereby rendering easier removal of        solvent after reaction. The switch from DMF to DCM was performed        for ease of scale-up as well as some reduced product yield that        was occasionally observed with reaction in DMF. The protection        of hydroxyl group with tert-butyldimethylsilyl chloride is        customarily done using DMF as solvent, although other solvents        have been used (Greene T W and Wuts P G M, Protecting Groups in        Organic Synthesis, John Wiley & Sons, Inc., 1999; pp 127-132).        In the context of SN-38, the use of DMF was preferred as the        material is only sparingly soluble in DCM. The high yield of the        product formation in DCM medium was unexpected and could not        have been anticipated.    -   3) In the conversion of 10-O-TBDMS-SN-38 to its chloroformate        reactive intermediate 5 in FIG. 1, triphosgene was added to the        dichloromethane solution in portions instead of one lot. This        preserved yield, while avoiding a potentially dangerous rise in        reaction temperatures in large-scale manufacture.    -   4) In the cycloaddition reaction between        azido-PEG-Lys(MMT)-PABO-CO-20-O-SN-38 (intermediate 7 in FIG. 1)        and MCC-Yne (intermediate 8 in FIG. 1) to result in the        penultimate intermediate, MCC-PEG-Lys(MMT)-PABOCO-20-O-SN-38        (intermediate 9 in FIG. 1), it was discovered that the product        was unstable if the reaction mixture was first worked up with        EDTA extraction to remove copper salt, leading to reduction in        yield. Surprisingly, it was also discovered that if the        chromatography of the reaction mixture was carried out first,        followed by extraction with EDTA to remove residual copper salt        that was not fully trapped in silica gel column, the product was        stable. This process change also led to improvements in yield by        avoiding the formation of unwanted side products. Improving the        stability of intermediate 9 prior to purification by simply        reversing the sequence of operations, namely performing        chromatography first followed by extraction with EDTA, was        unanticipated.    -   5) In the same reaction as described in item 4 above, improved        yield was obtained when the reaction time was reduced to 14 h,        instead of 18-24 h.

Additional improvements to the synthetic protocol are incorporated inFIG. 1, which were not disclosed in priority U.S. Pat. No. 9,107,960.These changes to the protocol, not obvious to one of ordinary skill inthe art, comprise the following changes.

-   -   6) The two-step conversion of starting material 1 to        intermediate 2 originally involved the isolation of the        intermediate from the first step. This process was telescoped        such that isolation of the intermediate between starting        material 1 and intermediate 2 was no longer necessary. Further        process improvements to this two-step conversion involved a        large reduction in the amount of volatile diethylamine used, a        safety improvement as well as facilitating its subsequent        removal during isolation of intermediate 2.    -   7) The two-step preparation of MCC-yne (intermediate 8), not        shown in the scheme, originally involved chromatographic        purification after each step; the reaction conditions were        changed for both steps, allowing isolation of both intermediate        8 and its precursor to be done by crystallization or        precipitation, without resorting to chromatography.    -   8) The two-step conversion of intermediate 3 to intermediate 7        originally involved the isolation of intermediate 6. This        process was simplified such that isolation of intermediate 6 was        no longer necessary. Further process improvements to this        two-step conversion included reduction in the number of phase        splits after each step (which resulted in increased yields and        reduction in the ensuant emulsions) as well as in the reduction        of the amount of TBAF required for the deprotection, resulting        in an improved purity profile. The chromatographic conditions        for isolation of intermediate 7 were also improved to ensure        adequate separation of impurities and full recovery of product        from the column.    -   9) The original conditions in the conversion of intermediate 7        to intermediate 9 involved the use of triphenylphosphine (PPh₃)        along with copper (I) bromide; these conditions contributed to        the slow degradation of intermediate 9 and so were replaced with        conditions [copper (II) sulfate, sodium ascorbate, and        2,6-lutidine] that provided much better solution stability of        intermediate 9 and much better overall quality of the final        product CL2A-SN-38

The changes to synthetic protocol described above resulted insubstantial improvements in yield and efficiency of production, as wellas decreased presence of contaminating biproducts in the final productand at various intermediate steps. The decrease in contaminants furtherallowed simplification or elimination of certain purification steps inthe intermediate synthetic reactions, also improving yield andefficiency.

After the CL2A-SN-38 product is formed, it may be conjugated to atargeting molecule, such as an antibody that binds to a tumor-associatedor other disease-associated antigen. In certain embodiments, when thebifunctional drug contains a thiol-reactive moiety as theantibody-binding group, the thiols on the antibody to be labeled may begenerated on the side chains of lysine groups of the antibody, insteadof reduced cysteine residues, by using a thiolating reagent. Methods forintroducing thiol groups onto antibodies by modifications of mAb lysinegroups are well known (Wong in Chemistry of protein conjugation andcross-linking, CRC Press, Inc., Boca Raton, Fla. (1991), pp 20-22).Alternatively, mild reduction of interchain disulfide bonds on theantibody using the reducing agent, tris-(2-carboxyethyl)phosphine(TCEP), can generate 7-to-10 thiols on the antibody; which has theadvantage of incorporating multiple drug moieties in the interchainregion of the MAb away from the antigen-binding region. Moreover, afterreduction with TCEP, a pre-purification of the reduced antibody is notnecessary, and the disulfide-reduced antibody is conjugated to thethiol-reactive CL2A-SN-38 in situ.

In another preferred embodiment, the conjugates of antibodies andCL2A-SN-38 are purified by tangential flow filtration (TFF) method usinga 50,000 Da molecular weight cut-off membrane using 25 to 30diafiltration volumes of the conjugate formulation buffer for purifyinghundreds of grams of the conjugates. This method obviates a need toemploy expensive and cumbersome chromatographic purifications onsize-exclusion and hydrophobic chromatography columns.

In yet another embodiment, the conjugates are formulated in Good'sbiological buffers at a pH of 6 to 7.0, and lyophilized for storage.Preferably, the Good's buffer is selected from the group consisting of2-(N-morpholino)ethanesulfonic acid (MES),3-(N-morpholino)propanesulfonic acid (MOPS),4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), and1,4-piperazinediethanesulfonic acid (PIPES), in the pH range of 6-7,preferably in the pH range of 6.5 to 7, and at a buffer concentration of10-100 mM, preferably 25 mM. The most preferred formulation buffer is 25mM IVIES, pH 6.5.

In further embodiment, the purified conjugates are combined withexcipients such as trehalose and polysorbate 80, lyophilized, and storedas lyophilates in the temperature range of −20° C. to 8° C.

General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures. Where antibodies of use in human therapy ordiagnosis are desired, the person of ordinary skill understand that thecorresponding human antigenic protein or peptide is preferably used toinduce antibody production.

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding Vκ (variable light chain) and V_(H) (variable heavychain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of an antibody from a cell thatexpresses a murine antibody can be cloned by PCR amplification andsequenced. To confirm their authenticity, the cloned V_(L) and V_(H)genes can be expressed in cell culture as a chimeric Ab as described byOrlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based onthe V gene sequences, a humanized antibody can then be designed andconstructed as described by Leung et al. (Mol. Immunol., 32: 1413(1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine antibody by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The Vκ sequence for the antibody may be amplified using theprimers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primerset described by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the Vκ andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human antibody. Alternatively, the Vκ andV_(H) expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990). In another embodiment, anantibody may be a human monoclonal antibody. Such antibodies may beobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge, asdiscussed below.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods. The skilled artisan will realize thatthis technique is exemplary only and any known method for making andscreening human antibodies or antibody fragments by phage display may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.). In this and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. Immunizationwith a target antigen will produce human antibodies by the normal immuneresponse, which may be harvested and/or produced by standard techniquesdiscussed above. A variety of strains of such transgenic mice areavailable, each of which is capable of producing a different class ofantibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the specific system, but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Production of Antibody Fragments

Antibody fragments may be obtained, for example, by pepsin or papaindigestion of whole antibodies by conventional methods. For example,antibody fragments may be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment maybe further cleaved using a thiol reducing agent and, optionally, ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages, to produce 3.5S Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using pepsin produces twomonovalent Fab fragments and an Fc fragment. Exemplary methods forproducing antibody fragments are disclosed in U.S. Pat. Nos. 4,036,945;U.S. Pat. No. 4,331,647; Nisonoff et al., 1960, Arch Biochem Biophys,89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al., 1967, METHODSIN ENZYMOLOGY, page 422 (Academic Press), and Coligan et al. (eds.),1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 06, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Other types of antibody fragments maycomprise one or more complementarity-determining regions (CDRs). CDRpeptides (“minimal recognition units”) can be obtained by constructinggenes encoding the CDR of an antibody of interest. Such genes areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region from RNA of antibody-producing cells. SeeLarrick et al., 1991, Methods: A Companion to Methods in Enzymology2:106; Ritter et al. (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (CambridgeUniversity Press); Birch et al., (eds.), 1995, MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.)

Antibody Variations

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797;each incorporated herein by reference).

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Stickler et al.,2011). It has been reported that G1m1 antibodies contain allotypicsequences that tend to induce an immune response when administered tonon-G1m1 (nG1m1) recipients, such as G1m3 patients (Stickler et al.,2011). Non-G1m1 allotype antibodies are not as immunogenic whenadministered to G1m1 patients (Stickler et al., 2011).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown belowfor the exemplary antibodies rituximab (SEQ ID NO:85) and veltuzumab(SEQ ID NO:86).

Rituximab heavy chain variable region sequence (SEQ ID NO: 85)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable region(SEQ ID NO: 86) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotypoe characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 1 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes 214 356/358 Complete allotype (allotype) (allotype)431 (allotype) Rituximab G1m17,1 K 17 D/L 1 A — Veltuzumab G1m3 R 3 E/M— A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Known Antibodies

In various embodiments, the claimed methods and compositions may utilizeany of a variety of antibodies known in the art. Antibodies of use maybe commercially obtained from a number of known sources. For example, avariety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.). A large numberof antibodies against various disease targets, including but not limitedto tumor-associated antigens, have been deposited at the ATCC and/orhave published variable region sequences and are available for use inthe claimed methods and compositions. See, e.g., U.S. Pat. Nos.7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310;6,444,206; 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;5,677,136; 5,587,459; 5,443,953; 5,525,338, the Examples section of eachof which is incorporated herein by reference. These are exemplary onlyand a wide variety of other antibodies and their hybridomas are known inthe art. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples sectionof each of which is incorporated herein by reference).

Particular antibodies that may be of use within the scope of the claimedmethods and compositions include, but are not limited to, LL1(anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20),rituxumab (anti-CD20), obinutuzumab (GA101, anti-CD20), lambrolizumab(anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab(anti-CTLA-4), RS7 (anti-epithelial glycoprotein-1 (EGP-1, also known asTROP-2)), PAM4 or KC4 (both anti-mucin), MN-14 (anti-carcinoembryonicantigen (CEA, also known as CD66e or CEACAM5), MN-15 or MN-3(anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu 31 (ananti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), IMMU-H2B(anti-H2B), IMMU-H3 (anti-H3), IMMU-H4 (anti-H4), TAG-72 (e.g., CC49),Tn, J591 or HuJ591 (anti-PSMA (prostate-specific membrane antigen)),AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (ananti-carbonic anhydrase IX MAb), L243 (anti-HLA-DR) alemtuzumab(anti-CD52), bevacizumab (anti-VEGF), cetuximab (anti-EGFR), gemtuzumab(anti-CD33), ibritumomab tiuxetan (anti-CD20); panitumumab (anti-EGFR);tositumomab (anti-CD20); PAM4 (aka clivatuzumab, anti-mucin) andtrastuzumab (anti-ErbB2). Such antibodies are known in the art (e.g.,U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393;6,653,104; 6,730,300; 6,899,864; 6,926,893; 6,962,702; 7,074,403;7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655;7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S. Patent ApplicationPubl. No. 20050271671; 20060193865; 20060210475; 20070087001; U.S.patent application Ser. No. 14/180,646; the Examples section of eachincorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,151,164),hA19 (U.S. Pat. No. 7,109,304), hIMMU-31 (U.S. Pat. No. 7,300,655), hLL1(U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No. 5,789,554), hMu-9 (U.S.Pat. No. 7,387,772), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat.No. 6,676,924), hMN-15 (U.S. Pat. No. 8,287,865), hR1 (U.S. patentapplication Ser. No. 13/688,812), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Other useful antigens that may be targeted using the describedconjugates include carbonic anhydrase IX, alpha-fetoprotein (AFP),α-actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733,BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCL19, CCL21, CD1,CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19,CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40,CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67,CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138,CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12,HIF-1α, colon-specific antigen-p (CSAp), CEACAM5, CEACAM6, c-Met, DAM,EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growthfactor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100,GRO-β, histone H2B, histone H3, histone H4, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxiainducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α,IFN-β, IFN-λ, IL -4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6,IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growthfactor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT,macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1,MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2,MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90,PAM4 antigen, pancreatic cancer mucin, PD-1, PD-L1, PD-1 receptor,placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA,PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE,S100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors,TNF-α, Tn antigen, Thomson-Friedenreich antigens, tumor necrosisantigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complementfactors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2, bcl-6,Kras, an oncogene marker and an oncogene product (see, e.g., Sensi etal., Clin Cancer Res 2006, 12:5023-32; Parmiani et al., J Immunol 2007,178:1975-79; Novellino et al. Cancer Immunol Immunother 2005,54:187-207).

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies fordrug-conjugated immunotherapy, is Craig and Foon, Blood prepublishedonline Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGM1 and CEA, respectively. These CD66antigens (e.g., CEACAM6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Penis, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., JControl Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8). Another useful target for breast cancer therapy is theLIV-1 antigen described by Taylor et al. (Biochem. J. 2003; 375:51-9).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases (Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009; Takahashiet al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is an exemplaryanti-CD74 antibody of therapeutic use for treatment of MIF-mediateddiseases.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

Checkpoint inhibitor antibodies have been used primarily in cancertherapy. Immune checkpoints refer to inhibitory pathways in the immunesystem that are responsible for maintaining self-tolerance andmodulating the degree of immune system response to minimize peripheraltissue damage. However, tumor cells can also activate immune systemcheckpoints to decrease the effectiveness of immune response againsttumor tissues. Exemplary checkpoint inhibitor antibodies againstcytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD152),programmed cell death protein 1 (PD1, also known as CD279) andprogrammed cell death 1 ligand 1 (PD-L1, also known as CD274), may beused in combination with one or more other agents to enhance theeffectiveness of immune response against disease cells, tissues orpathogens. Exemplary anti-PD1 antibodies include lambrolizumab (MK-3475,MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK),and pidilizumab (CT-011, CURETECH LTD.). Anti-PD1 antibodies arecommercially available, for example from ABCAM® (AB137132), BIOLEGEND®(EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE (J105, J116, MIH4).Exemplary anti-PD-L1 antibodies include MDX-1105 (MEDAREX), MEDI4736(MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB).Anti-PD-L1 antibodies are also commercially available, for example fromAFFYMETRIX EBIOSCIENCE (MIH1). Exemplary anti-CTLA4 antibodies includeipilimumab (Bristol-Myers Squibb) and tremelimumab (PFIZER). Anti-PD1antibodies are commercially available, for example from ABCAM®(AB134090), SINO BIOLOGICAL INC. (11159-H03H, 11159-H08H), and THERMOSCIENTIFIC PIERCE (PA5-29572, PA5-23967, PA5-26465, MA1-12205,MA1-35914). Ipilimumab has recently received FDA approval for treatmentof metastatic melanoma (Wada et al., 2013, J Transl Med 11:89).

In another preferred embodiment, antibodies are used that internalizerapidly and are then re-expressed, processed and presented on cellsurfaces, enabling continual uptake and accretion of circulatingconjugate by the cell. An example of a most-preferred antibody/antigenpair is LL1, an anti-CD74 MAb (invariant chain, class II-specificchaperone, Ii) (see, e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; theExamples section of each incorporated herein by reference). The CD74antigen is highly expressed on B-cell lymphomas (including multiplemyeloma) and leukemias, certain T-cell lymphomas, melanomas, colonic,lung, and renal cancers, glioblastomas, and certain other cancers (Onget al., Immunology 98:296-302 (1999)). A review of the use of CD74antibodies in cancer is contained in Stein et al., Clin Cancer Res. 2007Sep. 15; 13(18 Pt 2):5556s-5563s, incorporated herein by reference.

The diseases that are preferably treated with anti-CD74 antibodiesinclude, but are not limited to, non-Hodgkin's lymphoma, Hodgkin'sdisease, melanoma, lung, renal, colonic cancers, glioblastomemultiforme, histiocytomas, myeloid leukemias, and multiple myeloma.Continual expression of the CD74 antigen for short periods of time onthe surface of target cells, followed by internalization of the antigen,and re-expression of the antigen, enables the targeting LL1 antibody tobe internalized along with any chemotherapeutic moiety it carries. Thisallows a high, and therapeutic, concentration of LL1-chemotherapeuticdrug conjugate to be accumulated inside such cells. InternalizedLL1-chemotherapeutic drug conjugates are cycled through lysosomes andendosomes, and the chemotherapeutic moiety is released in an active formwithin the target cells.

In another preferred embodiment, the therapeutic conjugates can be usedagainst pathogens, since antibodies against pathogens are known. Forexample, antibodies and antibody fragments which specifically bindmarkers produced by or associated with infectious lesions, includingviral, bacterial, fungal and parasitic infections, for example caused bypathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi, andviruses, and antigens and products associated with such microorganismshave been disclosed, inter alia, in Hansen et al., U.S. Pat. No.3,927,193 and Goldenberg U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544,4,468,457, 4,444,744, 4,818,709 and 4,624,846, the Examples section ofeach incorporated herein by reference, and in Reichert and Dewitz, citedabove. A review listing antibodies against infectious organisms(antitoxin and antiviral antibodies), as well as other targets, iscontained in Casadevall, Clin Immunol 1999; 93(1):5-15, incorporatedherein by reference.

In a preferred embodiment, the pathogens are selected from the groupconsisting of HIV virus, Mycobacterium tuberculosis, Streptococcusagalactiae, methicillin-resistant Staphylococcus aureus, Legionellapneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseriagonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcusneoformans, Histoplasma capsulatum, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus,cytomegalovirus, herpes simplex virus I, herpes simplex virus II, humanserum parvo-like virus, respiratory syncytial virus, varicella-zostervirus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus,human T-cell leukemia viruses, Epstein-Barr virus, murine leukemiavirus, mumps virus, vesicular stomatitis virus, sindbis virus,lymphocytic choriomeningitis virus, wart virus, blue tongue virus,Sendai virus, feline leukemia virus, reovirus, polio virus, simian virus40, mouse mammary tumor virus, dengue virus, rubella virus, West Nilevirus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii,Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei,Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesiabovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica,Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis,Taenia saginata, Echinococcus granulosus, Mesocestoides corti,Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini,Acholeplasma laidlawii, M. salivarium and M. pneumoniae, as disclosed inU.S. Pat. No. 6,440,416, the Examples section of which is incorporatedherein by reference.

In a more preferred embodiment, drug conjugates of the present inventioncomprising anti-gp120 and other such anti-HIV antibodies can be used astherapeutics for HIV in AIDS patients; and drug conjugates of antibodiesto Mycobacterium tuberculosis are suitable as therapeutics fordrug-refractive tuberculosis. Fusion proteins of anti-gp120 MAb (antiHIV MAb) and a toxin, such as Pseudomonas exotoxin, have been examinedfor antiviral properties (Van Oigen et al., J Drug Target, 5:75-91,1998). Attempts at treating HIV infection in AIDS patients failed,possibly due to insufficient efficacy or unacceptable host toxicity. TheCPT drug conjugates of the present invention advantageously lack suchtoxic side effects of protein toxins, and are therefore advantageouslyused in treating HIV infection in AIDS patients. These drug conjugatescan be given alone or in combination with other antibiotics ortherapeutic agents that are effective in such patients when given alone.Candidate anti-HIV antibodies include the P4/D10 anti-envelope antibodydescribed by Johansson et al. (AIDS. 2006 Oct. 3; 20(15):1911-5), aswell as the anti-HIV antibodies described and sold by Polymun (Vienna,Austria), also described in U.S. Pat. No. 5,831,034, U.S. Pat. No.5,911,989, and Vcelar et al., AIDS 2007; 21(16):2161-2170 and Joos etal., Antimicrob. Agents Chemother. 2006; 50(5):1773-9, all incorporatedherein by reference. A preferred targeting agent for HIV is variouscombinations of these antibodies in order to overcome resistance.

Antibodies of use to treat autoimmune disease or immune systemdysfunctions (e.g., graft-versus-host disease, organ transplantrejection) are known in the art and may be conjugated to CL2A-SN-38using the disclosed methods and compositions. Antibodies of use to treatautoimmune/immune dysfunction disease may bind to exemplary antigensincluding, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4,CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20,CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43,CD45, CD55, TNF-alpha, interferon, IL-6 and HLA-DR. Antibodies that bindto these and other target antigens, discussed above, may be used totreat autoimmune or immune dysfunction diseases. Autoimmune diseasesthat may be treated with immunoconjugates may include acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, ANCA-associated vasculitides, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

The antibodies discussed above and other known antibodies againstdisease-associated antigens may be used as CL2A-SN-38-immunoconjugates,in the practice of the claimed methods and compositions.

Bispecific and Multispecific Antibodies

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). In certain embodiments, the techniques and compositionsfor therapeutic agent conjugation disclosed herein may be used withbispecific or multispecific antibodies as the antibody moieties.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez,et al. Nature. 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically crosslinking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. No.4,946,778 and U.S. Pat. No. 5,132,405, the Examples section of each ofwhich is incorporated herein by reference. Reduction of the peptidelinker length to less than 12 amino acid residues prevents pairing ofV_(H) and V_(L) domains on the same chain and forces pairing of V_(H)and V_(L) domains with complementary domains on other chains, resultingin the formation of functional multimers. Polypeptide chains of V_(H)and V_(L) domains that are joined with linkers between 3 and 12 aminoacid residues form predominantly dimers (termed diabodies). With linkersbetween 0 and 2 amino acid residues, trimers (termed triabody) andtetramers (termed tetrabody) are favored, but the exact patterns ofoligomerization appear to depend on the composition as well as theorientation of V-domains (V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), inaddition to the linker length.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, bispecific constructs known as “DOCK-AND-LOCK™” (DNL™)have been used to produce combinations of virtually any desiredantibodies, antibody fragments and other effector molecules (see, e.g.,U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and U.S. Ser.No. 11/925,408, the Examples section of each of which incorporatedherein by reference). The technique utilizes complementary proteinbinding domains, referred to as anchoring domains (AD) and dimerizationand docking domains (DDD), which bind to each other and allow theassembly of complex structures, ranging from dimers, trimers, tetramers,quintamers and hexamers. These form stable complexes in high yieldwithout requirement for extensive purification. The technique allows theassembly of monospecific, bispecific or multispecific antibodies. Any ofthe techniques known in the art for making bispecific or multispecificantibodies may be utilized in the practice of the presently claimedmethods.

Combinations of use, such as are preferred for cancer therapies, includeCD20+CD22 antibodies, CD74+CD20 antibodies, CD74+CD22 antibodies,CEACAM5 (CEA)+CEACAM6 (NCA) antibodies, insulin-like growth factor(ILGF)+CEACAM5 antibodies, EGP-1 (e.g., RS-7)+ILGF antibodies,CEACAM5+EGFR antibodies. Such antibodies need not only be used incombination, but can be combined as fusion proteins of various forms,such as IgG, Fab, scFv, and the like, as described in U.S. Pat. Nos.6,083,477; 6,183,744 and 6,962,702 and U.S. Patent ApplicationPublication Nos. 20030124058; 20030219433; 20040001825; 20040202666;20040219156; 20040219203; 20040235065; 20050002945; 20050014207;20050025709; 20050079184; 20050169926; 20050175582; 20050249738;20060014245 and 20060034759, the Examples section of each incorporatedherein by reference.

Pre-Targeting

Bispecific or multispecific antibodies may also be utilized inpre-targeting techniques. Pre-targeting is a multistep processoriginally developed to resolve the slow blood clearance of directlytargeting antibodies, which contributes to undesirable toxicity tonormal tissues such as bone marrow. With pre-targeting, a radionuclideor other therapeutic agent is attached to a small delivery molecule(targetable construct) that is cleared within minutes from the blood. Apre-targeting bispecific or multispecific antibody, which has bindingsites for the targetable construct as well as a target antigen, isadministered first, free antibody is allowed to clear from circulationand then the targetable construct is administered.

Pre-targeting methods are disclosed, for example, in Goodwin et al.,U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988;Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl.Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988;Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl.Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991;Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl.Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickney et al.,Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119, 1991; U.S.Pat. Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702;7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702; and6,962,702, each incorporated herein by reference.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antibody fragment; (2) optionally administeringto the subject a clearing composition, and allowing the composition toclear the antibody from circulation; and (3) administering to thesubject the targetable construct, containing one or more chelated orchemically bound therapeutic or diagnostic agents, such as SN-38.

Targetable Constructs

In certain embodiments, targetable construct peptides labeled with oneor more therapeutic or diagnostic agents for use in pre-targeting may beselected to bind to a bispecific antibody with one or more binding sitesfor a targetable construct peptide and one or more binding sites for atarget antigen associated with a disease or condition. Bispecificantibodies may be used in a pretargeting technique wherein the antibodymay be administered first to a subject. Sufficient time may be allowedfor the bispecific antibody to bind to a target antigen and for unboundantibody to clear from circulation. Then a targetable construct, such asa labeled peptide, may be administered to the subject and allowed tobind to the bispecific antibody and localize at the diseased cell ortissue.

Such targetable constructs can be of diverse structure and are selectednot only for the availability of an antibody or fragment that binds withhigh affinity to the targetable construct, but also for rapid in vivoclearance when used within the pre-targeting method and bispecificantibodies (bsAb) or multispecific antibodies. Hydrophobic agents arebest at eliciting strong immune responses, whereas hydrophilic agentsare preferred for rapid in vivo clearance. Thus, a balance betweenhydrophobic and hydrophilic character is established. This may beaccomplished, in part, by using hydrophilic chelating agents to offsetthe inherent hydrophobicity of many organic moieties. Also, sub-units ofthe targetable construct may be chosen which have opposite solutionproperties, for example, peptides, which contain amino acids, some ofwhich are hydrophobic and some of which are hydrophilic.

Peptides having as few as two amino acid residues, preferably two to tenresidues, may be used and may also be coupled to other moieties, such aschelating agents. The linker should be a low molecular weight conjugate,preferably having a molecular weight of less than 50,000 daltons, andadvantageously less than about 20,000 daltons, 10,000 daltons or 5,000daltons. More usually, the targetable construct peptide will have fouror more residues and one or more haptens for binding, e.g., to abispecific antibody. Exemplary haptens may include In-DTPA(indium-diethylene triamine pentaacetic acid) or HSG (histamine succinylglycine). The targetable construct may also comprise one or morechelating moieties, such as DOTA(1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid), NOTA(1,4,7-triaza-cyclononane-1,4,7-triacetic acid), TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid), NETA([2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-ethyl]-2-carbonylmethyl-amino]aceticacid) or other known chelating moieties. Chelating moieties may be used,for example, to bind to a therapeutic and or diagnostic radionuclide,paramagnetic ion or contrast agent.

The targetable construct may also comprise unnatural amino acids, e.g.,D-amino acids, in the backbone structure to increase the stability ofthe peptide in vivo. In alternative embodiments, other backbonestructures such as those constructed from non-natural amino acids orpeptoids may be used.

The peptides used as targetable constructs are conveniently synthesizedon an automated peptide synthesizer using a solid-phase support andstandard techniques of repetitive orthogonal deprotection and coupling.Free amino groups in the peptide, that are to be used later forconjugation of chelating moieties or other agents, are advantageouslyblocked with standard protecting groups such as a Boc group, whileN-terminal residues may be acetylated to increase serum stability. Suchprotecting groups are well known to the skilled artisan. See Greene andWuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons,N.Y.). When the peptides are prepared for later use within thebispecific antibody system, they are advantageously cleaved from theresins to generate the corresponding C-terminal amides, in order toinhibit in vivo carboxypeptidase activity.

Where pretargeting with bispecific antibodies is used, the antibody willcontain a first binding site for an antigen produced by or associatedwith a target tissue and a second binding site for a hapten on thetargetable construct. Exemplary haptens include, but are not limited to,HSG and In-DTPA. Antibodies raised to the HSG hapten are known (e.g. 679antibody) and can be easily incorporated into the appropriate bispecificantibody (see, e.g., U.S. Pat. Nos. 6,962,702; 7,138,103 and 7,300,644,incorporated herein by reference with respect to the Examples sections).However, other haptens and antibodies that bind to them are known in theart and may be used, such as In-DTPA and the 734 antibody (e.g., U.S.Pat. No. 7,534,431, the Examples section incorporated herein byreference).

Dock-and-Lock™ (DNL™)

In preferred embodiments, a bivalent or multivalent antibody is formedas a DOCK-AND-LOCK™ (DNL™) complex (see, e.g., U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examples section ofeach of which is incorporated herein by reference.) Generally, thetechnique takes advantage of the specific and high-affinity bindinginteractions that occur between a dimerization and docking domain (DDD)sequence of the regulatory (R) subunits of cAMP-dependent protein kinase(PKA) and an anchor domain (AD) sequence derived from any of a varietyof AKAP proteins (Baillie et al., FEBS Letters. 2005; 579: 3264. Wongand Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The DDD and ADpeptides may be attached to any protein, peptide or other molecule.Because the DDD sequences spontaneously dimerize and bind to the ADsequence, the technique allows the formation of complexes between anyselected molecules that may be attached to DDD or AD sequences.

Although the standard DNL™ complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL™complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL™ complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL™complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL™constructs of different stoichiometry may be produced and used (see,e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL™construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL™ constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1 (SEQ IDNO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4) CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL™ complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8) SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ (SEQ ID NO: 9)SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEE NRQILA PKA RIIα (SEQ IDNO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RIIβ (SEQ IDNO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:12 to SEQ ID NO:31 below. The skilled artisan will realizethat a large number of alternative species within the genus of DDDmoieties can be constructed by standard techniques, for example using acommercial peptide synthesizer or well known site-directed mutagenesistechniques. The effect of the amino acid substitutions on AD moietybinding may also be readily determined by standard binding assays, forexample as disclosed in Alto et al. (2003, Proc Natl Acad Sci USA100:4445-50).

TABLE 2 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1).Consensus sequence disclosed as SEQ ID NO: 87. S H I Q I P P G L T E L LQ G Y T V E V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F TR L R E A R A N N E D L D S K K D L K L I I I V V V

(SEQ ID NO: 12) THIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:13) SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14)SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 15)SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 16)SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 17)SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 18)SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 19)SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20)SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21)SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 22)SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23)SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24)SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 26)SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 27)SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 28)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 29)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 30)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 31)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 2 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:32 to SEQ ID NO:49 below. Again, a very large numberof species within the genus of possible AD moiety sequences could bemade, tested and used by the skilled artisan, based on the data of Altoet al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarger number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

TABLE 3 Conservative Amino Acid Substitutions in AD1 (SEQ ID NO: 3).Consensus sequence disclosed as SEQ ID NO: 88. Q I E Y L A K Q I V D N AI Q Q A N L D F I R N E Q N N L V T V I S V

(SEQ ID NO: 32) NIEYLAKQIVDNAIQQA (SEQ ID NO: 33) QLEYLAKQIVDNAIQQA (SEQID NO: 34) QVEYLAKQIVDNAIQQA (SEQ ID NO: 35) QIDYLAKQIVDNAIQQA (SEQ IDNO: 36) QIEFLAKQIVDNAIQQA (SEQ ID NO: 37) QIETLAKQIVDNAIQQA (SEQ ID NO:38) QIESLAKQIVDNAIQQA (SEQ ID NO: 39) QIEYIAKQIVDNAIQQA (SEQ ID NO: 40)QIEYVAKQIVDNAIQQA (SEQ ID NO: 41) QIEYLARQIVDNAIQQA (SEQ ID NO: 42)QIEYLAKNIVDNAIQQA (SEQ ID NO: 43) QIEYLAKQIVENAIQQA (SEQ ID NO: 44)QIEYLAKQIVDQAIQQA (SEQ ID NO: 45) QIEYLAKQIVDNAINQA (SEQ ID NO: 46)QIEYLAKQIVDNAIQNA (SEQ ID NO: 47) QIEYLAKQIVDNAIQQL (SEQ ID NO: 48)QIEYLAKQIVDNAIQQI (SEQ ID NO: 49) QIEYLAKQIVDNAIQQV

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:50),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL™ constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 50) QIEYVAKQIVDYAIHQA Alternative AKAPsequences (SEQ ID NO: 51) QIEYKAKQIVDHAIHQA (SEQ ID NO: 52)QIEYHAKQIVDHAIHQA (SEQ ID NO: 53) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs

AKAP-KL (SEQ ID NO: 54) PLEYQAGLLVQNAIQQAI AKAP79 (SEQ ID NO: 55)LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 56) LIEEAASRIVDAVIEQVK

RI-Specific AKAPs

AKAPce (SEQ ID NO: 57) ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 58)LEQVANQLADQIIKEAT PV38 (SEQ ID NO: 59) FEELAWKIAKMIWSDVF

Dual-Specificity AKAPs

AKAP7 (SEQ ID NO: 60) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 61)TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 62) QIKQAAFQLISQVILEAT DAKAP2 (SEQID NO: 63) LAWKIAKMIVSDVMQQ

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD (SEQ IDNO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 65)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 66) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 4 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 4 AKAP Peptide sequences Peptide Sequence AKAPIS QIEYLAKQIVDNAIQQA(SEQ ID NO: 3) AKAPIS-P QIEYLAKQIPDNAIQQA (SEQ ID NO: 67) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 5. The skilled artisan could readily derive alternative DDDamino acid sequences as disclosed above for Table 1 and Table 2.

TABLE 5 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1).Consensus sequence disclosed as SEQ ID NO: 89. S H I Q I P P G L T E L LQ G Y T V E V L R T N S I L A Q Q P P D L V E F A V E Y F T R L R E A RA N I D S K K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Alternative DNL™ Structures

In certain alternative embodiments, DNL™ constructs may be formed usingalternatively constructed antibodies or antibody fragments, in which anAD moiety may be attached at the C-terminal end of the kappa light chain(C_(k)), instead of the C-terminal end of the Fc on the heavy chain. Thealternatively formed DNL™ constructs may be prepared as disclosed inProvisional U.S. Patent Application Ser. No. 61/654,310, filed Jun. 1,2012, 61/662,086, filed Jun. 20, 2012, 61/673,553, filed Jul. 19, 2012,and 61/682,531, filed Aug. 13, 2012, the entire text of eachincorporated herein by reference. The light chain conjugated DNL™constructs exhibit enhanced Fc-effector function activity in vitro andimproved pharmacokinetics, stability and anti-lymphoma activity in vivo(Rossi et al., 2013, Bioconjug Chem 24:63-71).

C_(k)-conjugated DNL™ constructs may be prepared as disclosed inProvisional U.S. Patent Application Ser. No. 61/654,310, 61/662,086,61/673,553, and 61/682,531. Briefly, C_(k)-AD2-IgG, was generated byrecombinant engineering, whereby the AD2 peptide was fused to theC-terminal end of the kappa light chain. Because the natural C-terminusof C_(K) is a cysteine residue, which forms a disulfide bridge toC_(H)1, a 16-amino acid residue “hinge” linker was used to space the AD2from the C_(K)-V_(H)1 disulfide bridge. The mammalian expression vectorsfor C_(k)-AD2-IgG-veltuzumab and C_(k)-AD2-IgG-epratuzumab wereconstructed using the pdHL2 vector, which was used previously forexpression of the homologous C_(H)3-AD2-IgG modules. A 2208-bpnucleotide sequence was synthesized comprising the pdHL2 vector sequenceranging from the Bam HI restriction site within the V_(K)/C_(K) intronto the Xho I restriction site 3′ of the C_(k) intron, with the insertionof the coding sequence for the hinge linker (EFPKPSTPPGSSGGAP, SEQ IDNO:162) and AD2, in frame at the 3′end of the coding sequence for C_(K).This synthetic sequence was inserted into the IgG-pdHL2 expressionvectors for veltuzumab and epratuzumab via Bam HI and Xho I restrictionsites. Generation of production clones with SpESFX-10 were performed asdescribed for the C_(H)3-AD2-IgG modules. C_(k)-AD2-IgG-veltuzumab andC_(k)-AD2-IgG -epratuzumab were produced by stably-transfectedproduction clones in batch roller bottle culture, and purified from thesupernatant fluid in a single step using Mab Select (GE Healthcare)Protein A affinity chromatography.

Following the same DNL™ process described previously for 22-(20)-(20)(Rossi et al., 2009, Blood 113:6161-71), C_(k)-AD2-IgG-epratuzumab wasconjugated with C_(H)1-DDD2-Fab-veltuzumab, a Fab-based module derivedfrom veltuzumab, to generate the bsHexAb 22*-(20)-(20), where the 22*indicates the C_(k)-AD2 module of epratuzumab and each (20) symbolizes astabilized dimer of veltuzumab Fab. The properties of 22*-(20)-(20) werecompared with those of 22-(20)-(20), the homologous Fc-bsHexAbcomprising C_(H)3-AD2-IgG-epratuzumab, which has similar composition andmolecular size, but a different architecture.

Following the same DNL™ process described previously for 20-2b (Rossi etal., 2009, Blood 114:3864-71), C_(k)-AD2-IgG-veltuzumab, was conjugatedwith IFNα2b-DDD2, a module of IFNα2b with a DDD2 peptide fused at itsC-terminal end, to generate 20*-2b, which comprises veltuzumab with adimeric IFNα2b fused to each light chain. The properties of 20*-2b werecompared with those of 20-2b, which is the homologous Fc-IgG-IFNα.

Each of the bsHexAbs and IgG-IFNα were isolated from the DNL™ reactionmixture by MabSelect affinity chromatography. The two C_(k)-derivedprototypes, an anti-CD22/CD20 bispecific hexavalent antibody, comprisingepratuzumab (anti-CD22) and four Fabs of veltuzumab (anti-CD20), and aCD20-targeting immunocytokine, comprising veltuzumab and four moleculesof interferon-α2b, displayed enhanced Fc-effector functions in vitro, aswell as improved pharmacokinetics, stability and anti-lymphoma activityin vivo, compared to their Fc-derived counterparts.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL™ constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Phage Display

Certain embodiments of the claimed compositions and/or methods mayconcern binding peptides and/or peptide mimetics of various targetmolecules, cells or tissues. Binding peptides may be identified by anymethod known in the art, including but not limiting to the phage displaytechnique. Various methods of phage display and techniques for producingdiverse populations of peptides are well known in the art. For example,U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829 disclose methods forpreparing a phage library. The phage display technique involvesgenetically manipulating bacteriophage so that small peptides can beexpressed on their surface (Smith and Scott, 1985, Science228:1315-1317; Smith and Scott, 1993, Meth. Enzymol. 21:228-257). Inaddition to peptides, larger protein domains such as single-chainantibodies may also be displayed on the surface of phage particles (Arapet al., 1998, Science 279:377-380).

Targeting amino acid sequences selective for a given organ, tissue, celltype or target molecule may be isolated by panning (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). In brief, a library of phage containing putativetargeting peptides is administered to an intact organism or to isolatedorgans, tissues, cell types or target molecules and samples containingbound phage are collected. Phage that bind to a target may be elutedfrom a target organ, tissue, cell type or target molecule and thenamplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget organs, tissues, cell types or target molecule again andcollected for additional rounds of panning. Multiple rounds of panningmay be performed until a population of selective or specific binders isobtained. The amino acid sequence of the peptides may be determined bysequencing the DNA corresponding to the targeting peptide insert in thephage genome. The identified targeting peptide may then be produced as asynthetic peptide by standard protein chemistry techniques (Arap et al.,1998, Smith et al., 1985).

In some embodiments, a subtraction protocol may be used to furtherreduce background phage binding. The purpose of subtraction is to removephage from the library that bind to targets other than the target ofinterest. In alternative embodiments, the phage library may beprescreened against a control cell, tissue or organ. For example,tumor-binding peptides may be identified after prescreening a libraryagainst a control normal cell line. After subtraction the library may bescreened against the molecule, cell, tissue or organ of interest. Othermethods of subtraction protocols are known and may be used in thepractice of the claimed methods, for example as disclosed in U.S. Pat.Nos. 5,840,841, 5,705,610, 5,670,312 and 5,492,807.

Nanobodies

Nanobodies are single-domain antibodies of about 12-15 kDa in size(about 110 amino acids in length). Nanobodies can selectively bind totarget antigens, like full-size antibodies, and have similar affinitiesfor antigens. However, because of their much smaller size, they may becapable of better penetration into solid tumors. The smaller size alsocontributes to the stability of the nanobody, which is more resistant topH and temperature extremes than full size antibodies (Van Der Linden etal., 1999, Biochim Biophys Act 1431:37-46). Single-domain antibodieswere originally developed following the discovery that camelids (camels,alpacas, llamas) possess fully functional antibodies without lightchains (e.g., Hamsen et al., 2007, Appl Microbiol Biotechnol 77:13-22).The heavy-chain antibodies consist of a single variable domain (V_(HH))and two constant domains (C_(H)2 and C_(H)3). Like antibodies,nanobodies may be developed and used as multivalent and/or bispecificconstructs. Humanized forms of nanobodies are in commercial developmentthat are targeted to a variety of target antigens, such as IL-6R, vWF,TNF, RSV, RANKL, IL-17A & F and IgE (e.g., ABLYNX®, Ghent, Belgium),with potential clinical use in cancer, inflammation, infectious disease,Alzheimer's disease, acute coronary syndrome and other disorders (e.g.,Saerens et al., 2008, Curr Opin Pharmacol 8:600-8; Muyldermans, 2013,Ann Rev Biochem 82:775-97; Ibanez et al., 2011, J Infect Dis203:1063-72).

The plasma half-life of nanobodies is shorter than that of full-sizeantibodies, with elimination primarily by the renal route. Because theylack an Fc region, they do not exhibit complement dependentcytotoxicity.

Nanobodies may be produced by immunization of camels, llamas, alpacas orsharks with target antigen, following by isolation of mRNA, cloning intolibraries and screening for antigen binding. Nanobody sequences may behumanized by standard techniques (e.g., Jones et al., 1986, Nature 321:522, Riechmann et al., 1988, Nature 332: 323, Verhoeyen et al., 1988,Science 239: 1534, Carter et al., 1992, Proc. Nat'l Acad. Sci. USA 89:4285, Sandhu, 1992, Crit. Rev. Biotech. 12: 437, Singer et al., 1993, J.Immun. 150: 2844). Humanization is relatively straight-forward becauseof the high homology between camelid and human FR sequences.

In various embodiments, the subject CL2A-SN-38 conjugates may comprisenanobodies for targeted delivery of conjugated drug to cells, tissues,organs or pathogens. Nanobodies of use are disclosed, for example, inU.S. Pat. Nos. 7,807,162; 7,939,277; 8,188,223; 8,217,140; 8,372,398;8,557,965; 8,623,361 and 8,629,244, the Examples section of eachincorporated herein by reference.)

Conjugation Protocols

The preferred conjugation protocol is based on a thiol-maleimide, athiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamidereaction that are facile at neutral or acidic pH. This obviates the needfor higher pH conditions for conjugations as, for instance, would benecessitated when using active esters. Further details of exemplaryconjugation protocols are described below in the Examples section.

Therapeutic Treatment

In another aspect, the invention relates to a method of treating asubject, comprising administering a therapeutically effective amount ofa therapeutic conjugate as described herein to a subject. Diseases thatmay be treated with the therapeutic conjugates described herein include,but are not limited to B-cell malignancies (e.g., non-Hodgkin'slymphoma, mantle cell lymphoma, multiple myeloma, Hodgkin's lymphoma,diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma,acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cellleukemia) using, for example an anti-CD22 antibody such as the hLL2 MAb(epratuzumab, see U.S. Pat. No. 6,183,744), against another CD22 epitope(hRFB4) or antibodies against other B cell antigens, such as CD19, CD20,CD21, CD22, CD23, CD37, CD40, CD40L, CD52, CD74, CD80 or HLA-DR. Otherdiseases include, but are not limited to, adenocarcinomas ofendodermally-derived digestive system epithelia, cancers such as breastcancer and non-small cell lung cancer, and other carcinomas, sarcomas,glial tumors, myeloid leukemias, etc. In particular, antibodies againstan antigen, e.g., an oncofetal antigen, produced by or associated with amalignant solid tumor or hematopoietic neoplasm, e.g., agastrointestinal, stomach, colon, esophageal, liver, lung, breast,pancreatic, liver, prostate, ovarian, testicular, brain, bone orlymphatic tumor, a sarcoma or a melanoma, are advantageously used. Suchtherapeutics can be given once or repeatedly, depending on the diseasestate and tolerability of the conjugate, and can also be used optionallyin combination with other therapeutic modalities, such as surgery,external radiation, radioimmunotherapy, immunotherapy, chemotherapy,antisense therapy, interference RNA therapy, gene therapy, and the like.Each combination will be adapted to the tumor type, stage, patientcondition and prior therapy, and other factors considered by themanaging physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term. Dosesgiven herein are for humans, but can be adjusted to the size of othermammals, as well as children, in accordance with weight or square metersize.

In a preferred embodiment, therapeutic conjugates comprising ananti-EGP-1 (anti-TROP-2) antibody such as the hRS7 MAb can be used totreat carcinomas such as carcinomas of the esophagus, pancreas, lung,stomach, colon and rectum, urinary bladder, breast, ovary, uterus,kidney and prostate, as disclosed in U.S. Pat. Nos. 7,238,785; 7,517,964and 8,084,583, the Examples section of which is incorporated herein byreference. An hRS7 antibody is a humanized antibody that comprises lightchain complementarity-determining region (CDR) sequences CDR1(KASQDVSIAVA, SEQ ID NO:90); CDR2 (SASYRYT, SEQ ID NO:91); and CDR3(QQHYITPLT, SEQ ID NO:92) and heavy chain CDR sequences CDR1 (NYGMN, SEQID NO:93); CDR2 (WINTYTGEPTYTDDFKG, SEQ ID NO:94) and CDR3(GGFGSSYWYFDV, SEQ ID NO:95)

In another preferred embodiment, therapeutic conjugates comprising ananti-CEACAM5 antibody (e.g., hMN-14, labretuzumab) and/or ananti-CEACAM6 antibody (e.g., hMN-3 or hMN-15) may be used to treat anyof a variety of cancers that express CEACAM5 and/or CEACAM6, asdisclosed in U.S. Pat. Nos. 7,541,440; 7,951,369; 5,874,540; 6,676,924and 8,267,865, the Examples section of each incorporated herein byreference. Solid tumors that may be treated using anti-CEACAM5,anti-CEACAM6, or a combination of the two include but are not limited tobreast, lung, pancreatic, esophageal, medullary thyroid, ovarian, colon,rectum, urinary bladder, mouth and stomach cancers. A majority ofcarcinomas, including gastrointestinal, respiratory, genitourinary andbreast cancers express CEACAM5 and may be treated with the subjectimmunoconjugates. An hMN-14 antibody is a humanized antibody thatcomprises light chain variable region CDR sequences CDR1 (KASQDVGTSVA;SEQ ID NO:96), CDR2 (WTSTRIIT; SEQ ID NO:97), and CDR3 (QQYSLYRS; SEQ IDNO:98), and the heavy chain variable region CDR sequences CDR1 (TYWMS;SEQ ID NO:99), CDR2 (EIHPDSSTINYAPSLKD; SEQ ID NO:100) and CDR3(LYFGFPWFAY; SEQ ID NO:101).

An hMN-3 antibody is a humanized antibody that comprises light chainvariable region CDR sequences CDR1 (RSSQSIVHSNGNTYLE, SEQ ID NO:102),CDR2 (KVSNRFS, SEQ ID NO:103) and CDR3 (FQGSHVPPT, SEQ ID NO:104) andthe heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:105), CDR2(WINTYTGEPTYADDFKG, SEQ ID NO:106) and CDR3 (KGWMDFNSSLDY, SEQ IDNO:107).

An hMN-15 antibody is a humanized antibody that comprises light chainvariable region CDR sequences SASSRVSYIH (SEQ ID NO:108); GTSTLAS (SEQID NO:109); and QQWSYNPPT (SEQ ID NO:110); and heavy chain variableregion CDR sequences DYYMS (SEQ ID NO:111); FIANKANGHTTDYSPSVKG (SEQ IDNO:112); and DMGIRWNFDV (SEQ ID NO:113).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD74 antibody (e.g., hLL1, milatuzumab, disclosed in U.S. Pat. Nos.7,074,403; 7,312,318; 7,772,373; 7,919,087 and 7,931,903, the Examplessection of each incorporated herein by reference) may be used to treatany of a variety of cancers that express CD74, including but not limitedto renal, lung, intestinal, stomach, breast, prostate or ovarian cancer,as well as several hematological cancers, such as multiple myeloma,chronic lymphocytic leukemia, acute lymphoblastic leukemia, non-Hodgkinlymphoma, and Hodgkin lymphoma. An hLL1 antibody is a humanized antibodycomprising the light chain CDR sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ IDNO:114), CDR2 (TVSNRFS; SEQ ID NO:115), and CDR3 (SQSSHVPPT; SEQ IDNO:116) and the heavy chain variable region CDR sequences CDR1 (NYGVN;SEQ ID NO:117), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO:118), and CDR3(SRGKNEAWFAY; SEQ ID NO:119).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD22 antibody (e.g., hLL2, epratuzumab, disclosed in U.S. Pat. Nos.5,789,554; 6,183,744; 6,187,287; 6,306,393; 7,074,403 and 7,641,901, theExamples section of each incorporated herein by reference, or thechimeric or humanized RFB4 antibody) may be used to treat any of avariety of cancers that express CD22, including but not limited toindolent forms of B-cell lymphomas, aggressive forms of B-celllymphomas, chronic lymphatic leukemias, acute lymphatic leukemias,non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt lymphoma, follicularlymphoma or diffuse B-cell lymphoma. Anti-CD22 conjugates are also ofuse to treat autoimmune diseases, such as acute immune thrombocytopenia,chronic immune thrombocytopenia, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, pemphigusvulgaris, diabetes mellitus (e.g., juvenile diabetes), Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, ANCA-associated vasculitides, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis, fibrosingalveolitis, graft-versus-host disease (GVHD), organ transplantrejection, sepsis, septicemia and inflammation. An hLL2 antibody is ahumanized antibody comprising light chain CDR sequences CDR1(KSSQSVLYSANHKYLA, SEQ ID NO:120), CDR2 (WASTRES, SEQ ID NO:121), andCDR3 (HQYLSSWTF, SEQ ID NO:122) and the heavy chain CDR sequences CDR1(SYWLH, SEQ ID NO:123), CDR2 (YINPRNDYTEYNQNFKD, SEQ ID NO:124), andCDR3 (RDITTFY, SEQ ID NO:125)

In a preferred embodiment, therapeutic conjugates comprising anti-CSApantibodies, such as the hMu-9 MAb, can be used to treat colorectal, aswell as pancreatic and ovarian cancers as disclosed in U.S. Pat. Nos.6,962,702; 7,387,772; 7,414,121; 7,553,953; 7,641,891 and 7,670,804, theExamples section of each incorporated herein by reference. An hMu-9antibody is a humanized antibody comprising light chain CDR sequencesCDR1 (RSSQSIVHSNGNTYLE, SEQ ID NO:126), CDR2 (KVSNRFS, SEQ ID NO:127),and CDR3 (FQGSRVPYT, SEQ ID NO:128), and heavy chain variable CDRsequences CDR1 (EYVIT, SEQ ID NO:129), CDR2 (EIYPGSGSTSYNEKFK, SEQ IDNO:130), and CDR3 (EDL, SEQ ID NO:131).

Therapeutic conjugates comprising the hPAM4 MAb can be used to treatpancreatic cancer or other solid tumors, as disclosed in U.S. Pat. Nos.7,238,786 and 7,282,567, the Examples section of each incorporatedherein by reference. An hPAM4 antibody is a humanized antibodycomprising light chain variable region CDR sequences CDR1 (SASSSVSSSYLY,SEQ ID NO:132); CDR2 (STSNLAS, SEQ ID NO:133); and CDR3 (HQWNRYPYT, SEQID NO:134); and heavy chain CDR sequences CDR1 (SYVLH, SEQ ID NO:135);CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:136) and CDR3 (GFGGSYGFAY, SEQ IDNO:137).

In another preferred embodiment, therapeutic conjugates comprising ananti-AFP MAb, such as IMMU31, can be used to treat hepatocellularcarcinoma, germ cell tumors, and other AFP-producing tumors usinghumanized, chimeric and human antibody forms, as disclosed in U.S. Pat.No. 7,300,655, the Examples section of which is incorporated herein byreference. An IMMU31 antibody is a humanized antibody comprising theheavy chain CDR sequences CDR1 (SYVIH, SEQ ID NO:138), CDR2(YIHPYNGGTKYNEKFKG, SEQ ID NO:139) and CDR3 (SGGGDPFAY, SEQ ID NO:140)and the light chain CDR1 (KASQDINKYIG, SEQ ID NO:141), CDR2 (YTSALLP,SEQ ID NO:142) and CDR3 (LQYDDLWT, SEQ ID NO:143).

In another preferred embodiment, therapeutic conjugates comprising ananti-HLA-DR MAb, such as hL243, can be used to treat lymphoma, leukemia,cancers of the skin, esophagus, stomach, colon, rectum, pancreas, lung,breast, ovary, bladder, endometrium, cervix, testes, kidney, liver,melanoma or other HLA-DR-producing tumors, as disclosed in U.S. Pat. No.7,612,180, the Examples section of which is incorporated herein byreference. An hL243 antibody is a humanized antibody comprising theheavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:144), CDR2(WINTYTREPTYADDFKG, SEQ ID NO:145), and CDR3 (DITAVVPTGFDY, SEQ IDNO:146) and light chain CDR sequences CDR1 (RASENIYSNLA, SEQ ID NO:147),CDR2 (AASNLAD, SEQ ID NO:148), and CDR3 (QHFWTTPWA, SEQ ID NO:149).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD20 MAb, such as veltuzumab (hA20), 1F5, obinutuzumab (GA101), orrituximab, can be used to treat lymphoma, leukemia, immunethrombocytopenic purpura, systemic lupus erythematosus, Sjögren'ssyndrome, Evans syndrome, arthritis, arteritis, pemphigus vulgaris,renal graft rejection, cardiac graft rejection, rheumatoid arthritis,Burkitt lymphoma, non-Hodgkin's lymphoma, follicular lymphoma, smalllymphocytic lymphoma, diffuse B-cell lymphoma, marginal zone lymphoma,chronic lymphocytic leukemia, acute lymphocytic leukemia, Type Idiabetes mellitus, GVHD, multiple sclerosis or multiple myeloma, asdisclosed in U.S. Pat. No. 7,435,803 or 8,287,864, the Examples sectionof each incorporated herein by reference. An hA20 (veltuzumab) antibodyis a humanized antibody comprising the light chain CDR sequences CDRL1(RASSSVSYIH, SEQ ID NO:150), CDRL2 (ATSNLAS, SEQ ID NO:151) and CDRL3(QQWTSNPPT, SEQ ID NO:152) and heavy chain CDR sequences CDRH1 (SYNMH,SEQ ID NO:153), CDRH2 (AIYPGNGDTSYNQKFKG, SEQ ID NO:154) and CDRH3(STYYGGDWYFDV, SEQ ID NO:155).

In another preferred embodiment, therapeutic conjugates comprising ananti-CD19 MAb, such as hA19, can be used to treat B-cell relatedlymphomas and leukemias, such as non-Hodgkin's lymphoma, chroniclymphocytic leukemia or acute lymphoblastic leukemia. Other diseasestates that may be treated include autoimmune diseases, such as acute orchronic immune thrombocytopenia, dermatomyositis, Sydenham's chorea,myasthenia gravis, systemic lupus erythematosus, lupus nephritis,rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetesmellitus, Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosurn, Takayasu's arteritis, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis ubiterans, Sjögren'ssyndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis, and fibrosingalveolitis, as disclosed in U.S. Pat. Nos. 7,109,304, 7,462,352,7,902,338, 8,147,831 and 8,337,840, the Examples section of eachincorporated herein by reference. An hA19 antibody is a humanizedantibody comprising the light chain CDR sequences CDR1 KASQSVDYDGDSYLN(SEQ ID NO: 156); CDR2 DASNLVS (SEQ ID NO: 157); and CDR3 QQSTEDPWT (SEQID NO: 158) and the heavy chain CDR sequences CDR1 SYWMN (SEQ ID NO:159); CDR2 QIWPGDGDTNYNGKFKG (SEQ ID NO: 160) and CDR3 RETTTVGRYYYAMDY(SEQ ID NO: 161).

Therapeutic conjugates comprising anti-tenascin antibodies can be usedto treat hematopoietic and solid tumors, and conjugates comprisingantibodies to tenascin can be used to treat solid tumors, preferablybrain cancers like glioblastomas.

Preferably, the antibodies that are used in the treatment of humandisease are human or humanized (CDR-grafted) versions of antibodies;although murine and chimeric versions of antibodies can be used. Samespecies IgG molecules as delivery agents are mostly preferred tominimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Antibodies such as hLL1 and hLL2 rapidly internalize afterbinding to internalizing antigen on target cells, which means that thechemotherapeutic drug being carried is rapidly internalized into cellsas well. However, antibodies that have slower rates of internalizationcan also be used to effect selective therapy.

The therapeutic conjugates can be used against pathogens, sinceantibodies against pathogens are known. For example, antibodies andantibody fragments which specifically bind markers produced by orassociated with infectious lesions, including viral, bacterial, fungaland parasitic infections, for example caused by pathogens such asbacteria, rickettsia, mycoplasma, protozoa, fungi, and viruses, andantigens and products associated with such microorganisms have beendisclosed, inter alia, in Hansen et al., U.S. Pat. No. 3,927,193 andGoldenberg U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457,4,444,744, 4,818,709 and 4,624,846, the Examples section of eachincorporated herein by reference, and in Reichert and Dewitz, citedabove. In a preferred embodiment, the pathogens are selected from thegroup consisting of HIV virus, Mycobacterium tuberculosis, Streptococcusagalactiae, methicillin-resistant Staphylococcus aureus, Legionellapneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseriagonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcusneoformans, Histoplasma capsulatum, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus,cytomegalovirus, herpes simplex virus I, herpes simplex virus II, humanserum parvo-like virus, respiratory syncytial virus, varicella-zostervirus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus,human T-cell leukemia viruses, Epstein-Barr virus, murine leukemiavirus, mumps virus, vesicular stomatitis virus, sindbis virus,lymphocytic choriomeningitis virus, wart virus, blue tongue virus,Sendai virus, feline leukemia virus, reovirus, polio virus, simian virus40, mouse mammary tumor virus, dengue virus, rubella virus, West Nilevirus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii,Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei,Trypanosoma brucei, Schistosoma mansoni, Schistosoma japanicum, Babesiabovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica,Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis,Taenia saginata, Echinococcus granulosus, Mesocestoides corti,Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini,Acholeplasma laidlawii, M. salivarium and M. pneumoniae, as disclosed inU.S. Pat. No. 6,440,416, the Examples section of which is incorporatedherein by reference.

Drug conjugates of the present invention comprising anti-gp120 and othersuch anti-HIV antibodies can be used as therapeutics for HIV in AIDSpatients; and drug conjugates of antibodies to Mycobacteriumtuberculosis are suitable as therapeutics for drug-refractivetuberculosis. Fusion proteins of anti-gp120 MAb (anti HIV MAb) and atoxin, such as Pseudomonas exotoxin, have been examined for antiviralproperties (Van Oigen et al., J Drug Target, 5:75-91, 1998). Attempts attreating HIV infection in AIDS patients failed, possibly due toinsufficient efficacy or unacceptable host toxicity. The drug conjugatesof the present invention advantageously lack such toxic side effects ofprotein toxins, and are therefore advantageously used in treating HIVinfection in AIDS patients. These drug conjugates can be given alone orin combination with other antibiotics or therapeutic agents that areeffective in such patients when given alone. Candidate anti-HIVantibodies include the P4/D10 anti-envelope antibody described byJohansson et al. (AIDS. 2006 Oct. 3; 20(15):1911-5), as well as theanti-HIV antibodies described and sold by Polymun (Vienna, Austria),also described in U.S. Pat. No. 5,831,034, U.S. Pat. No. 5,911,989, andVcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob.Agents Chemother. 2006; 50(5):1773-9, all incorporated herein byreference. A preferred targeting agent for HIV is various combinationsof these antibodies in order to overcome resistance.

A more effective incorporation into cells and pathogens can beaccomplished by using multivalent, multispecific or multivalent,monospecific antibodies. Examples of such bivalent and bispecificantibodies are found in U.S. Pat. Nos. 7,387,772; 7,300,655; 7,238,785;and 7,282,567, the Examples section of each of which is incorporatedherein by reference. These multivalent or multispecific antibodies areparticularly preferred in the targeting of cancers and infectiousorganisms (pathogens), which express multiple antigen targets and evenmultiple epitopes of the same antigen target, but which often evadeantibody targeting and sufficient binding for immunotherapy because ofinsufficient expression or availability of a single antigen target onthe cell or pathogen. By targeting multiple antigens or epitopes, saidantibodies show a higher binding and residence time on the target, thusaffording a higher saturation with the drug being targeted in thisinvention.

The therapeutic conjugates may also be used to treat autoimmune diseaseor immune system dysfunction (e.g., graft-versus-host disease, organtransplant rejection). Antibodies of use to treat autoimmune/immunedysfunction disease may bind to exemplary antigens including, but notlimited to, BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4, CD5, CD7, CD8,CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20, CD21, CD22,CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43, CD45, CD55,CD56, CCD57, CD59, CD64, CD71, CD74, CD79a, CD79b, CD117, CD138, FMC-7,H2B, H3, H4, HLA-DR and MIF. Antibodies that bind to these and othertarget antigens, discussed above, may be used to treat autoimmune orimmune dysfunction diseases. Autoimmune diseases that may be treatedwith immunoconjugates may include acute idiopathic thrombocytopenicpurpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

The person of ordinary skill will realize that the subjectimmunoconjugates, comprising a camptothecin conjugated to an antibody orantibody fragment, may be used alone or in combination with one or moreother therapeutic agents, such as a second antibody, second antibodyfragment, second immunoconjugate, radionuclide, toxin, drug,chemotherapeutic agent, radiation therapy, chemokine, cytokine,immunomodulator, enzyme, hormone, oligonucleotide, RNAi or siRNA. Suchadditional therapeutic agents may be administered separately, incombination with, or attached to the subject antibody-drugimmunoconjugates.

In certain embodiments, a therapeutic agent used in combination with thecamptothecin conjugate of this invention may comprise one or moreisotopes. Radioactive isotopes useful for treating diseased tissueinclude, but are not limited to—¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu,⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, ²²⁷Th and ²¹¹Pb. The therapeutic radionuclide preferably has adecay-energy in the range of 20 to 6,000 keV, preferably in the ranges60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter,and 4,000-6,000 keV for an alpha emitter. Maximum decay energies ofuseful beta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213, Th-227 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Radionuclides and other metals may be delivered, for example, usingchelating groups attached to an antibody or conjugate. Macrocyclicchelates such as NOTA, DOTA, and TETA are of use with a variety ofmetals and radiometals, most particularly with radionuclides of gallium,yttrium and copper, respectively. Such metal-chelate complexes can bemade very stable by tailoring the ring size to the metal of interest.Other ring-type chelates, such as macrocyclic polyethers for complexing²²³Ra, may be used.

Therapeutic agents of use in combination with the camptothecinconjugates described herein also include, for example, chemotherapeuticdrugs such as vinca alkaloids, anthracyclines, epidophyllotoxins,taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents,antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic andproapoptotic agents, particularly doxorubicin, methotrexate, taxol,other camptothecins, and others from these and other classes ofanticancer agents, and the like. Other cancer chemotherapeutic drugsinclude nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, pyrimidine analogs, purine analogs, platinumcoordination complexes, hormones, and the like. Suitablechemotherapeutic agents are described in REMINGTON'S PHARMACEUTICALSCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN ANDGILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillanPublishing Co. 1985), as well as revised editions of these publications.Other suitable chemotherapeutic agents, such as experimental drugs, areknown to those of skill in the art.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib,AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine,dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, flavopiridol,fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine,hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib,L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839. Such agents may bepart of the conjugates described herein or may alternatively beadministered in combination with the described conjugates, either priorto, simultaneously with or after the conjugate. Alternatively, one ormore therapeutic naked antibodies as are known in the art may be used incombination with the described conjugates. Exemplary therapeutic nakedantibodies are described above.

Therapeutic agents that may be used in concert with the camptothecinconjugates also may comprise toxins conjugated to targeting moieties.Toxins that may be used in this regard include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. (See, e.g., Pastan. et al., Cell (1986), 47:641,and Sharkey and Goldenberg, CA Cancer J Clin. 2006 July-August;56(4):226-43.) Additional toxins suitable for use herein are known tothose of skill in the art and are disclosed in U.S. Pat. No. 6,077,499.

Yet another class of therapeutic agent may comprise one or moreimmunomodulators. Immunomodulators of use may be selected from acytokine, a stem cell growth factor, a lymphotoxin, an hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF), interferon,such as interferons-α, -β, -γ or -λ, and stem cell growth factor, suchas that designated “S1 factor”. Included among the cytokines are growthhormones such as human growth hormone, N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, -γ and -λ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand lymphotoxin (LT). As used herein, the term cytokine includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

Formulation and Administration

Suitable routes of administration of the conjugates include, withoutlimitation, oral, parenteral, rectal, transmucosal, intestinaladministration, intramedullary, intrathecal, direct intraventricular,intravenous, or intraperitoneal injections. The preferred routes ofadministration are parenteral. Alternatively, one may administer thecompound in a local rather than systemic manner, for example, viainjection of the compound directly into a solid tumor.

Immunoconjugates can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the immunoconjugate iscombined in a mixture with a pharmaceutically suitable excipient.Sterile phosphate-buffered saline is one example of a pharmaceuticallysuitable excipient. Other suitable excipients are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof

In a preferred embodiment, the immunoconjugate is formulated in Good'sbiological buffer (pH 6-7), using a buffer selected from the groupconsisting of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (IVIES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes]. More preferredbuffers are IVIES or MOPS, preferably in the concentration range of 20to 100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/vpolysorbate 80 as excipients, with the final buffer concentrationmodified to 22.25 mM as a result of added excipients. The preferredmethod of storage is as a lyophilized formulation of the conjugates,stored in the temperature range of −20° C. to 2° C., with the mostpreferred storage at 2° C. to 8° C.

The immunoconjugate can be formulated for intravenous administrationvia, for example, bolus injection, slow infusion or continuous infusion.Preferably, the antibody of the present invention is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours. For example, the first 25-50 mg could beinfused within 30 minutes, preferably even 15 min, and the remainderinfused over the next 2-3 hrs. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the immunoconjugate. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan immunoconjugate from such a matrix depends upon the molecular weightof the immunoconjugate, the amount of immunoconjugate within the matrix,and the size of dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989); Sherwood et al., supra. Other solid dosage forms aredescribed in Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

Generally, the dosage of an administered immunoconjugate for humans willvary depending upon such factors as the patient's age, weight, height,sex, general medical condition and previous medical history. It may bedesirable to provide the recipient with a dosage of immunoconjugate thatis in the range of from about 1 mg/kg to 24 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. A dosage of 1-20 mg/kg for a 70kg patient, for example, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-mpatient. The dosage may be repeated as needed, for example, once perweek for 4-10 weeks, once per week for 8 weeks, or once per week for 4weeks. It may also be given less frequently, such as every other weekfor several months, or monthly or quarterly for many months, as neededin a maintenance therapy. Preferred dosages may include, but are notlimited to, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22mg/kg and 24 mg/kg. Any amount in the range of 1 to 24 mg/kg may beused. The dosage is preferably administered multiple times, once ortwice a week. A minimum dosage schedule of 4 weeks, more preferably 8weeks, more preferably 16 weeks or longer may be used. The schedule ofadministration may comprise administration once or twice a week, on acycle selected from the group consisting of: (i) weekly; (ii) everyother week; (iii) one week of therapy followed by two, three or fourweeks off; (iv) two weeks of therapy followed by one, two, three or fourweeks off; (v) three weeks of therapy followed by one, two, three, fouror five week off; (vi) four weeks of therapy followed by one, two,three, four or five week off; (vii) five weeks of therapy followed byone, two, three, four or five week off; and (viii) monthly. The cyclemay be repeated 4, 6, 8, 10, 12, 16 or 20 times or more.

Alternatively, an immunoconjugate may be administered as one dosageevery 2 or 3 weeks, repeated for a total of at least 3 dosages. Or,twice per week for 4-6 weeks. If the dosage is lowered to approximately200-300 mg/m² (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a70 kg patient), it may be administered once or even twice weekly for 4to 10 weeks. Alternatively, the dosage schedule may be decreased, namelyevery 2 or 3 weeks for 2-3 months. It has been determined, however, thateven higher doses, such as 12 mg/kg once weekly or once every 2-3 weekscan be administered by slow i.v. infusion, for repeated dosing cycles.The dosing schedule can optionally be repeated at other intervals anddosage may be given through various parenteral routes, with appropriateadjustment of the dose and schedule

In preferred embodiments, the immunoconjugates are of use for therapy ofcancer. Examples of cancers include, but are not limited to, carcinoma,lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, orlymphoid malignancies. More particular examples of such cancers arenoted below and include: squamous cell cancer (e.g., epithelial squamouscell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, gastric or stomach cancer including gastrointestinalcancer, pancreatic cancer, glioblastoma multiforme, cervical cancer,ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellularcarcinoma, neuroendocrine tumors, medullary thyroid cancer,differentiated thyroid carcinoma, breast cancer, ovarian cancer, coloncancer, rectal cancer, endometrial cancer or uterine carcinoma, salivarygland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer,anal carcinoma, penile carcinoma, as well as head-and-neck cancer. Theterm “cancer” includes primary malignant cells or tumors (e.g., thosewhose cells have not migrated to sites in the subject's body other thanthe site of the original malignancy or tumor) and secondary malignantcells or tumors (e.g., those arising from metastasis, the migration ofmalignant cells or tumor cells to secondary sites that are differentfrom the site of the original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenström's macroglobulinemia, Wilms' tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias; e.g., acute lymphocytic leukemia, acute myelocytic leukemia[including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia]) and chronic leukemias (e.g., chronic myelocytic[granulocytic] leukemia and chronic lymphocytic leukemia), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Autoimmune diseases that may be treated with immunoconjugates mayinclude acute and chronic immune thrombocytopenias, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one conjugated antibody or other targeting moiety as describedherein. If the composition containing components for administration isnot formulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

General

Abbreviations used below are: DCC, dicyclohexylcarbodiimide; NHS,N-hydroxysuccinimide, DMAP, 4-dimethylaminopyridine; EEDQ,2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; MMT, monomethoxytrityl;PABOH, p-aminobenzyl alcohol; PEG, polyethylene glycol; SMCC,succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; TBAF,tetrabutylammonium fluoride; TBDMS, tert-butyldimethylsilyl chloride.

Chloroformates of hydroxy compounds in the following examples wereprepared using triphosgene and DMAP according to the procedure describedin Moon et al. (J. Medicinal Chem. 51:6916-6926, 2008), which isincorporated by reference. Extractive work-up refers to extraction withchloroform, dichloromethane or ethyl acetate, and washing optionallywith saturated bicarbonate, water, and with saturated sodium chloride.Flash chromatography was done using 230-400 mesh silica gel andmethanol-dichloromethane gradient, using up to 15% v/vmethanol-dichloromethane, unless otherwise stated. Reverse phase HPLCwas performed by Method A using a 7.8×300 mm C18 HPLC column, fittedwith a precolumn filter, and using a solvent gradient of 100% solvent Ato 100% solvent B in 10 minutes at a flow rate of 3 mL per minute andmaintaining at 100% solvent B at a flow rate of 4.5 mL per minute for 5or 10 minutes; or by Method B using a 4.6×30 mm Xbridge C18, 2.5 μm,column, fitted with a precolumn filter, using the solvent gradient of100% solvent A to 100% of solvent B at a flow rate of 1.5 mL per minutesfor 4 min and 100% of solvent B at a flow rate of 2 mL per minutes for 1minutes. Solvent A was 0.3% aqueous ammonium acetate, pH 4.46 whilesolvent B was 9:1 acetonitrile-aqueous ammonium acetate (0.3%), pH 4.46.HPLC was monitored by a dual in-line absorbance detector set at 360 nmand 254 nm.

Example 1 Preparation of CL2A-SN-38

A preferred reaction scheme for synthesis of CL2A-SN-38 is shown inFIG. 1. A previous version of the synthetic protocol, described in U.S.Pat. No. 9,107,960, was the basis for the improved protocol shown inFIG. 1. The substrates, intermediates and reactions disclosed in U.S.Pat. No. 9,107,960 are discussed in the present Example. Furtherimprovements to the protocol of U.S. Pat. No. 9,107,960, incorporated inFIG. 1, are described in the following Examples.

Preparation ofO-(2-Azidoethyl)-O′—[(N-diglycolyl-2-aminoethyl)-Lys(MMT)-PABOH]heptaethyleneglycol(intermediate 3): In a 500-mL single-neck flask, commercially availableFmoc-Lys(MMT)-OH (16 g), p-aminobenzyl alcohol (3.26 g) and EEDQ (6.52g) were added, followed by anhydrous dichloromethane (80 mL). Afterstirring overnight, diethylamine (25 mL) was added, and after a further6 h, the reaction mixture was concentrated to a volume of ˜50 mL. Thiswas diluted with heptane, and the solution was concentrated back to 50mL. Two additional chases with heptane (50 mL each) provided a biphasicmixture containing gummy material at the bottom. The gummy material wastaken up in dichloromethane (24 mL), stirred, and treated to a slowaddition of heptane (80 mL). After stirring for 1 h, the slurry wasfiltered to obtain 13.02 g (99.6% yield) of Lys(MMT)-PABOH (intermediate2, FIG. 1). The material was further optionally purified bychromatography if residual diethylamine was found to be present.Lys(MMT)-PABOH (11.51 g) was mixed with a solution ofO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol(PEG-N₃; 12.107 g) in anhydrous dichloromethane (90 mL). To this stirredsolution was added EEDQ (5.54 g). After˜18 h, the reaction mixture wasconcentrated and purified by chromatography on silica gel, using ethylacetate-methanol gradient elution, to obtain pure title product(intermediate 3, FIG. 1).

Preparation of TBDMS-SN-38 (intermediate 4): The preparation wasmodified with the use of dichloromethane, instead of dimethylformamide,as solvent, which enabled easier work up. A solution ofdiisopropylethylamine (36.6 g) in anhydrous dichloromethane (160 mL) wasadded to SN-38 (33 g). The suspension was cooled in ice/water bath, andstirred. To this stirred suspension was added a solution ofter-butyldimethylsilyl chloride (31.72 g) in dichloromethane (125 mL).The cold bath was removed, and the reaction mixture was stirred for 4 h.The clear reaction mixture was washed with 380 mL of 0.2 M HCl in 10%sodium chloride solution and 300 mL of 10% sodium chloride solution. Theproduct, after drying and solvent removal, was precipitated from ethylacetate-heptane to obtain 38.75 g (91% yield) of TBDMS-SN-38.

Generation of 10-O-TBDMS-SN-38-20-O-chloroformate (reactive intermediate5) and preparation ofO-(2-azidoethyl)-O′—[(N-diglycolyl-2-aminoethyl)-Lys(MMT)-PABOCO-20-O-SN-38]heptaethyleneglycol(intermediate 7): To avoid large exotherm from the bolus addition of therequired amounts of triphosgene, improved process was invented by addinga solution of triphosgene (0.24 g) in anhydrous dichloromethane (4 mL),over 30 minutes, to a stirred mixture of 0.94 g of TBDMS-SN-38 and DMAP(0.76 g) in dichloromethane (17 mL). This resulted in 98.5% conversionto a reactive intermediate (5 in FIG. 1), as determined by HPLC analysisof a quenched aliquot of the reaction mixture, quenched with anhydrousmethanol. The clean formation of the required product, without theevidence of the formation of a dimeric material due to quenching ofinitially formed chloroformate with unreacted SN-38, was thus assured.While bolus addition resulted in a large exotherm of 5.8° C. to 17.6°C., the slow addition maintained the internal temperature at <10° C.throughout, while not compromising the quality of the reaction. The sameapproach was repeated using 11.25 g (22.2 mmol) of TBDMS-SN-38 in 250 mLof anhydrous dichloromethane, and using other reagents proportionatelyas described for the small-scale reaction above. After 15 min, asolution of 25.01 g ofO-(2-Azidoethyl)-O′—[(N-diglycolyl-2-aminoethyl)-Lys(MMT)-PABOH]heptaethyleneglycol(intermediate 3, FIG. 1) in dichloromethane (125 mL) was added over 8min. After 75 min, the reaction mixture was sequentially washed with 50mM sodium acetate buffer, pH 5.3, water, and brine, and dried overanhydrous sodium sulfate. The solution of the product was then stirredwith a mixture of 1 M tetrabutylammonium fluoride in tetrahydrofuran (31mL), anhydrous dichloromethane (34 mL) and acetic acid (3 mL). After 2h, the reaction mixture was washed with 0.25 M citrate buffer, pH 6,water, and brine, and dried over anhydrous sodium sulfate.Chromatography on silica gel, with gradient elution withmethanol-methylene chloride mixtures furnished 17.3 g (52% yield) of thetitle product (intermediate 7).

Preparation of CL2A-SN-38:O-(2-azidoethyl)-O′—[(N-diglycolyl-2-aminoethyl)-Lys(MMT)-PABOCO-20-O-SN-38]heptaethyleneglycol(intermediate 7, 10.2 g) in anhydrous dichloromethane (130 mL) was mixedwith 4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide(intermediate 8 [‘MCC-yne’]; 3.78 g). To this, a mixture oftriphenylphosphine (0.38 g), cuprous bromide (0.24 g), anddiisorpropylethylamine (0.25 mL) in dichloromethane (100 mL) was added.The reaction mixture was stirred at ambient room temperature for 14 h.The material was concentrated and purified by chromatography; the pureproduct solution was washed with EDTA, water, and brine. The product,O-{2-[(1,2,3-triazolyl)-4-[4-(N-maleimidomethyl)cyclohexane-1-carboxamidomethyl]]ethyl}-O′—[(N-diglycolyl-2-aminoethyl)-Lys(MMT)-PABOCO-20-O-SN-38]heptaethyleneglycol(intermediate 9) was obtained in the amount of 10.8 g (89% yield), whichwas better than the maximum yield of 67% obtained previously. Thismodified procedure improved the yield because of reduced reaction time(14 h versus 18-41 h), and also avoided a previously encounteredstability problem with intermediate 9 by performing chromatography firstand EDTA extraction subsequently. The product solution indichloromethane was mixed with anisole (2.8 g), cooled to <5° C., andreacted with dichloroacetic acid (5.8 g) for 2 h. The final product,CL2A-SN-38, was precipitated from tert-butyl methyl ether in an overallyield (in 2 steps) of 81%.

Example 2 Improved Protocol for Synthesis of CL2A-SN-38

While the synthetic protocol disclosed in Example 1 above is suitablefor large-scale preparation of CL2A-SN-38, further improvements aredisclosed herein that were not known and could not have been predictedas of the filing date of U.S. Pat. No. 9,107,960.

The CL2A linker disclosed herein comprises a PEG moiety containing adefined number of PEG monomers. As shown in FIG. 1, in a preferredembodiment the number of PEG monomers used in the linker is eight.However, alternative embodiments within the scope of the invention mayutilize a number of PEG monomers between 1-30, more preferably 1-12,more preferably 6-10, more preferably 8.

There are 3 primary stages in which process improvements were made: (a)in the conversion of intermediate 6 to intermediate 7; (b) in theconversion of intermediate 8 to intermediate 9; and (c) in theconversion of intermediate 9 to intermediate 10.

Yield Improvement in the Preparation of Intermediate 7

In the conversion of silyl-protected intermediate 6 to desilylatedintermediate 7, tetrabutylammonium fluoride used was changed from1.4-fold molar excess in previous versions to 1.1-fold molar excess inthe present protocol. This level of the reagent was equally efficient,and also reduced the impurity profile, thereby enhancing the purityprofile of intermediate 7 and leading to improved yield.

The aqueous work-ups in the preparations of both intermediates 6 and 7were reduced as follows. In previous versions, the work-up involvedwashing organic extracts four times with 0.05 M sodium acetate buffer,pH 5.3 for intermediate 6 and four times with 0.25 M citrate buffer, pH6 for intermediate 7, with water washes subsequently in each case. Inimproved protocol, the buffer washes were reduced to 3 and 2 forintermediates 6 and 7, respectively, and the water washes were removedin both cases. These modifications considerably reduced the formation ofemulsion, and improved recovery of product in the organic extract.

The chromatographic purification of intermediate 7 previously involvedthe use of dichloromethane-methanol mixtures for elution. In theimproved protocol of FIG. 1, the elution was modified to a combinationof dichloromethane-ethyl acetate-methanol mixtures with varying methanolconcentration, followed by varying concentration ofdichloromethane-methanol mixtures. This resulted in a better and afaster separation of the intermediate, and improved the recovery. Theratios of solvents used are discussed in more detail in Example 4 below.

The overall yield in the 3-step sequence from intermediate 4 tointermediate 7 was improved from the 29-40% range to 64% byincorporating these process changes.

Improvement in Process Robustness in the Preparation of Intermediate 9

In the previously disclosed protocol(s), the copper (+1)-catalyzedreaction between intermediates 7 and 8 to produce intermediate 9 wasperformed using a freshly-formed complex of cuprous bromide andtriphenylphosphine in dichloromethane with a tertiary amine, such asdiisopropylethyl amine, added. This procedure worked well, producingintermediate 9 in yields of 50-60% after purification. However, theratio of cuprous bromide and triphenylphosphine in the complex needed tobe carefully controlled. It was shown that in presence oftriphenylphosphine alone, the product quality of the intermediate 9deteriorated over time, indicating the deleterious effect of thisreagent, which required a quick removal of this reagent once thecopper-catalyzed reaction was completed. This introduces a certainlimitation, with potential to cause reduction in the yield of purifiedintermediate 9, particularly in large-scale preparations.

In the modified procedure disclosed herein, the reaction was performedusing a bi-phasic mixture containing copper sulfate and ascorbic acid inwater and intermediates 7 and 8, as well as 2,6-collidine, indichloromethane. By stirring the biphasic solution overnight, typically18 h, the conversion was complete, and the crude product was separatedfrom copper sulfate and ascorbate by a simple wash with aqueous EDTA andpurified by chromatography. The product yield was similar to that withcuprous bromide/triphenylphosphine as catalyst, but the improvedprotocol avoids use of triphenylphosphine and its possible effects onstability of intermediates.

Process Improvement in the Preparation of Final Product 10 (CL2A-SN-38)

The stability profile of intermediate 9 in solution and as isolatedmaterial without solvent showed that the product maintained purityof >97% either stored as solid product at −20° C. for 4 days or assolution in dichloromethane at room temperature for 1 day. In the lattercase, storage at room temperature for 4 days reduced the purity to89.4%, while the solution stored at −20° C. showed a purity of only 92%.In the previously disclosed protocol, the material in dichloromethane,procured after chromatography and EDTA wash, was used as is for thedeprotection reaction mediated by dichloroacetic acid (DCA) and anisole.In that situation, depending on the length of storage of theintermediate 9 in solution form, the quality of the final material(CL2A-SN-38) varied. In the improved protocol, intermediate 9, afterpurification, was concentrated to a solid that was subsequently used inthe final deprotection step. This modification removed uncertainty aboutchanges in product quality of intermediate 9 prior to the finaldeprotection step.

Another improvement related to adding the solution of crude product,after the reaction, dropwise over several hours, into vigorously stirredtert-butyl methyl ether (t-BME), which enabled the precipitation of theproduct in the form of filterable solid. If the addition of the reactionmixture is not gradual and if the stirring is not vigorous, the productoiled out instead of precipitating as a solid.

Other changes may be incorporated into various steps in the syntheticprotocol of FIG. 1. For example, in the conversion of intermediate 1 tointermediate 2 and in the conversion of intermediate 2 to intermediate3, EEDQ is shown as amide coupling reagent. However, the person ofordinary skill will realize that amide coupling may be accomplishedusing numerous alternative reagents, as reviewed in: Han S-Y and KimY-A. Recent development of peptide coupling reagents in organicsynthesis. Tetrahedron 2004; 60: 2447-2467; and Valleur E and Bradley M.Amide bond formation: beyond the myth of coupling reagents. Chem Soc Rev2009; 38:606-631. In non-limiting examples, useful amide couplingreagents may be selected from: (1) carbodiimide reagents such asdicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), or1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) used in conjunctionwith additives selected from 1-hydroxy-1H-benzotriazole (HOBt),N-hydroxysuccinimide (HOSu), and dimethylaminopyridine (DMAP); (2)uronium salts O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) andO-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU); (3)N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) andN-isobutoxycarbonyl-2-isobutoxy-1,2-dihydroquinoline (IIDQ); and (4)isobutylchloroformate.

Example 3 Use of 1.1 vs. 1.4 Equivalents of Tetrabutylammonium Fluoride(TBAF) for Desilylation of Intermediate 6 to Intermediate 7

The crude intermediate 6 (13.78 g; 0.873 mmol) dissolved in 14 mL of DCMwas cooled and treated with 1.4 equiv of 1 M tetrabutylammonium fluoride(TBAF) containing acetic acid (0.126 g), with the reaction mixturemaintained at ≤20° C. After 1 h, the reaction mixture was washed with0.25 M citrate buffer, pH 6 (3×10 mL), water, and 5% sodium chloridesolution, and dried over anhydrous sodium sulfate. HPLC showed 85.5%purity for the crude product 7, with impurities at four peak positionsas follows: 1.0% (relative retention time or RRT of 0.79), 2.4% (RRT of0.864), 1.5% (RRT of 0.94) and 3.7% (RRT of 1.162). When the samereaction was conducted using 1.1 equiv of 1 M TBAF, HPLC purity of theproduct improved slightly to 88.2%, but the impurities droppedsignificantly: 0.3% (RRT of 0.79), 0.7% (RRT of 0.864), 0.8% (RRT of0.94) and 2.4% (RRT of 1.162). Improvement in these impurity profilessimplified the subsequent chromatographic separation.

Example 4 Use of Ternary Solvent System Vs. Binary Solvent System in theChromatography of Intermediate 7

A solution of 17.2 g of crude intermediate 7 was applied to a silica gelcolumn packed in mobile phase A (MPA), which was 15% dichloromethane(DCM)/ethyl acetate. Elution was performed by step gradient using 0-5%methanol-MPA, followed by a 2:1 mixture of 5% methanol-MPA and 5%methanol-DCM; a 1:2 mixture of 5% methanol/MPA and 5% methanol-DCM; andfinally 5% methanol/DCM. Seven fractions collected showed a productpurity of >93%, resulting in a yield of 69.6% for the product versus44.7% yield for the previously disclosed binary chromatographic elutionsystem. In the latter system, a solution of 17.2 g of crude intermediate7 was applied to a silica gel column under the same conditions as forthe modified procedure, but with elution by step gradient using 0-5%methanol-DCM mixtures. Product with a purity of >93% was obtained onlyin 2 fractions, resulting in a yield of 44.7%. In subsequentexperiments, the MPA was made with 30% DCM/ethyl acetate, which was thenmixed with varying methanol concentrations for elution.

Example 5 Steps to Improve Yield in the 3-step Conversion ofIntermediate 4 to Intermediate 7

A solution of 8.868 g (17.5 mmol) of TBDMS-SN-38 (intermediate 3) and4-[N,N -dimethylamino]pyridine (DMAP; 7.06 g; 3.3 equiv) in 133 mL ofdichloromethane (DCM) was stirred under nitrogen, and cooled to aninternal temperature of 2.6° C. To this solution was added, over 6minutes, a solution of triphosgene (2.4 g; 8.088 mmol) in 44 mL of DCM.The reaction mixture was warmed to an internal temperature of 15° C.over 20 minutes and kept at 15-16° C. for 10 min. The reaction mixturewas cooled to an internal temperature of 6.4° C., followed by theaddition of a solution of intermediate 3 (18.03 mmol, 1.03 equiv) in97.5 mL of anhydrous DCM, while maintaining the internal temperature at≤15° C. The reaction mixture was stirred for 3.5 h, and washed with 50mM sodium acetate, pH 5.3, buffer (3×97.5 g) and 10% sodium chloride(97.5 g), and dried over anhydrous sodium sulfate overnight.

The filtered solution was cooled to an internal temperature of ≤15° C.,and treated with a solution of 1 M TBAF (19.3 mL, 19.3 mmol, 1.1 equiv)in 27 mL of DCM containing acetic acid (2.52 g, 42.0 mmol, 2.4 equiv).The mixture was stirred at 10-16° C. for 45 min, and then washed with2×300 g of a mixture of 400 g of 0.25 M citrate buffer, pH 6, and 200 gof 10% sodium chloride and dried over anhydrous sodium sulfate. Thedried solution was concentrated to ˜50 mL and applied to a column of 400g of silica gel packed in 30% DCM/ethyl acetate (MPA). Elution wascarried out with a step gradient containing an increasing concentrationof methanol (1-5% methanol in MPA), followed by elution with 5% methanolin 2:1 MPA/DCM, 5% methanol in 1:2 MPA/DCM, and 5% methanol/DCM. Thepurest fractions, with purity 94%, were combined, concentrated to ˜50mL, chased with ethyl acetate (EtOAc; 2×80 mL) and EtOAC/heptane (1:1;160 mL) to a final volume of 35-50 mL, and diluted with 160 mL ofheptane. The precipitated solid product was filtered and dried at 40° C.under vacuum. The yield was 17.21 g (64.3% after adjusting for residual3.2 wt % heptane). The HPLC purity of the product, intermediate 7, was95%. ¹H NMR spectrum confirmed the structure. The isolated yield of64.3% was higher than the 29-40% obtained with previously disclosedprotocols.

Example 6 Stability of Intermediate 9 in the Presence ofTriphenylphosphine

Pure, combined, chromatographic fractions of intermediate 9 wereevaluated in the presence of 0 mol % (control), 10 mol %, and 20 mol %of triphenylphosphine at room temperature over a period of 96 h. Table 6below shows that the product quality deteriorated in presence oftriphenylphosphine.

TABLE 6 % HPLC purity of intermediate 9 (solution form) in the presenceof triphenylphosphine (Ph₃P) at room temperature Sample 0 h 6 h 26 h 96h Intermediate 9 + 96.0 % 94.2 % 93.7 % 91.6 % No Ph₃P (0 mol %)Intermediate 9 + Not 88.2 % 84.8 % 85.5 % Ph₃P (10 mol %) availableIntermediate 9 + 71.6 % 73.0 % 67.5 % 68.8 % Ph₃P (20 mol %)

Example 7 Improvements for Preparing Intermediate 9

An amber bottle was charged with intermediate 7 (n=8; 232 g; 157 mmol; 1equiv) and methylene chloride (2.1 kg) to form a solution. The solutionwas transferred to a 12-L round-bottom flask equipped with an overheadstirrer, temperature probe, and nitrogen blanket. To the round bottomflask was charged intermediate 8 (77 g, 280 mmol, 1.8 equiv), methylenechloride (1.2 kg), 1 M copper (II) sulfate solution (32 g), 1M(+)-sodium L-ascorbate solution (93 g), and 2,6-lutidine (20 g). Theresultant solution was stirred overnight. After 18.5 h, the reactionmixture was washed six times with 1.5 L of 0.1 M EDTA solution, pH 5.5.The organic layer was concentrated to 0.7 L and purified by flashchromatography on 6 kg of silica gel packed on an 11″ wide column.Elution was carried out with 0-8% methanol-methylene chloride mixturesin volumes of 5.8, 14.5, 14.5, 23.2, 23.2, 23.2, 23.2, and 46.4 litersfor successive step-gradients. The pure fractions were combined and thesolvents were evaporated. Recovery: 145 g (52.6% yield; HPLC purity:96.5%).

Example 8 Stability Profile of Intermediate 9

Intermediate 9 was prepared using a biphasic mixture of aqueous coppersulfate/sodium ascorbate and DCM solution of intermediates 7 and 8.Product isolation gave a 60-65% yield of pure intermediate 9. Bothsolvent-free product (solid product) and combined pure fractions fromchromatography (solution form) were subjected to stability analysis.Table 7 gives stability data by HPLC analysis. From the data, it wasdetermined that intermediate 9 is best stored in solid form at lowtemperature prior to deprotection to CL2A-SN-38.

TABLE 7 % HPLC purity of intermediate 9 stored under various conditionsSolution form: Solution form: Solution form: Solid product: HPLC Peak−20° C., 4 days Room temp., 1 day Room temp., 4 days −20° C., 4 daysInt. 9 (RRT 1.0) 92.0% 97.8% 89.4% 97.3% IMP (RRT 0.396) 2.4%   0% 2.7%0.6% IMP (RRT 0.805) 4.9%  2.2% 6.4% 1.2% IMP: impurity peak; RRT:relative retention time on HPLC, with RRT for intermediate 9 taken as 1.

Example 9 Preparation of CL2A-SN-38 (Final Product 10)

Intermediate 9 (145 g) was dissolved in methylene chloride (1.7 kg), andthe solution was transferred to a 5-L round-bottom flask equipped withan overhead stirrer, temperature probe, and nitrogen blanket. To thesolution was added anisole (39 g; 359 mmol, 4.35 equiv), and thesolution was cooled to 4° C. Dichloroacetic acid (107 g, 831 mmol, 10.08equiv) was added over 20 min, maintaining the temperature at ≤4° C.Following addition, the temperature was increased to 15° C., and thereaction mixture was stirred for 4 h. This reaction mixture was added totert-butyl methyl ether (t-BME; 3.8 kg), taken in a 22-L round-bottomflask fitted with an overhead stirrer, addition funnel, and nitrogeninlet, over 1.5 h, while maintaining vigorous mixing. The resultingslurry was stirred at room temperature for 12 h. The solids were allowedto settle, and the supernatant was decanted. The solids were rinsed with4% methylene chloride/t-BME, slurried with t-BME and filtered. Thecollected solids were dried at ≤40° C. under vacuum to obtain 124.3 g ofthe title compound as yellow solid (93.6% yield).

Example 10 Conjugation of CL2A-SN-38 to Antibodies

The anti-CEACAM5 humanized MAb, hMN-14, the anti-CD22 humanized MAb,hLL2, the anti-CD20 humanized MAb, hA20, the anti-EGP-1 humanized MAb,hRS7, and anti-mucin humanized MAb, hPAM4, were used in these studies.Each antibody was mildly reduced with Tris(2-carboxyethyl)phosphine(TCEP) in phosphate buffer at pH in the range of 7-7.4, the pH wasadjusted to 6.5, and reacted with ˜10-fold molar excess of CL2A-SN-38using DMSO at 5-10% v/v as co-solvent, and incubating for 20 min atambient temperature. Any excess thiol was capped with N-ethylmaleimideused as an aqueous solution at a 10-fold molar excess with respect toantibody.

The conjugate was purified by tangential flow filtration (TFF), using20-30 diafiltration volumes of the final formulation buffer, 25 mM MES,pH 6.5. This method avoided cumbersome sequential purification onsize-exclusion and hydrophobic columns, thereby enabling hundreds ofgrams of conjugates to be purified in a facile manner. The product wasassayed for SN-38 by absorbance at 366 nm and correlating with standardvalues. The protein concentration was deduced from absorbance at 280 nm,corrected for spillover of SN-38 absorbance at this wavelength. Fromthese, the SN-38/MAb substitution ratios (DAR) were determined. Thepurified conjugates were stored as lyophilized formulations in glassvials, capped under vacuum and stored in a −20° C. freezer. DARsobtained for some of these conjugates, which were typically in the5-to-7 range, are shown in Table 8.

TABLE 8 SN-38/MAb Drug/MAb ratios (DAR) in some conjugates MAb ConjugateDAR hMN-14 hMN-14-[CL2A-SN-38] 6.1 hRS7 hRS7-CL2A-SN-38 5.8 hA20hA20-CL2A-SN-38 5.8 hLL2 hLL2-CL2A-SN-38 5.7 hPAM4 hPAM4-CL2A-SN-38 5.9

Example 11 In Vivo Therapeutic Efficacies in Preclinical Models of HumanPancreatic or Colon Carcinoma

Immune-compromised athymic female nude mice, bearing subcutaneous humanpancreatic or colon tumor xenografts were treated with either specificCL2A-SN-38 conjugate or control conjugate or were left untreated. Thetherapeutic efficacies of the specific conjugates were observed. FIG. 2shows a Capan 1 pancreatic tumor model, wherein specific CL2A-SN-38conjugates of hRS7 (anti-EGP-1), hPAM4 (anti-mucin), and hMN-14(anti-CEACAM5) antibodies showed better efficacies than controlhA20-CL2A-SN-38 conjugate (anti-CD20) and untreated control. Similarlyin a BXPC3 model of human pancreatic cancer, the specifichRS7-CL2A-SN-38 showed better therapeutic efficacy than controltreatments (FIG. 3). Likewise, in an aggressive LS174T model of humancolon carcinoma, treatment with specific hMN-14-CL2A-SN-38 was moreefficacious than non-treatment (FIG. 4).

Example 12 Use of Humanized Anti-TROP-2 IgG-SN-38 Conjugate forEffective Treatment of Diverse Epithelial Cancers

The purpose of this study was to evaluate the efficacy of anSN-38-anti-TROP-2 antibody-drug conjugate (ADC) against several humansolid tumor types, and to assess its tolerability in mice and monkeys,the latter with tissue cross-reactivity to hRS7 similar to humans. TwoSN-38 derivatives, CL2-SN-38 (see U.S. Pat. No. 7,591,994) andCL2A-SN-38, were conjugated to the anti-TROP-2-humanized antibody, hRS7.The immunoconjugates were characterized in vitro for stability, binding,and cytotoxicity. Efficacy was tested in five different human solidtumor-xenograft models that expressed TROP-2 antigen. Toxicity wasassessed in mice and in Cynomolgus monkeys.

The hRS7 conjugates of the two SN-38 derivatives were equivalent in drugsubstitution (˜6), cell binding (K_(d)˜1.2 nmol/L), cytotoxicity(IC₅₀˜2.2 nmol/L), and serum stability in vitro (t/_(1/2)˜20 hours).Exposure of cells to the ADC demonstrated signaling pathways leading toPARP cleavage, but differences versus free SN-38 in p53 and p21upregulation were noted. Significant antitumor effects were produced byhRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P≤0.05), Capan-1(P<0.018), BxPC-3 (P<0.005), and COLO 205 tumors (P<0.033) when comparedto nontargeting control ADCs. Mice tolerated a dose of 2×12 mg/kg (SN-38equivalents) with only short-lived elevations in ALT and AST liverenzyme levels. Cynomolgus monkeys infused with 2×0.96 mg/kg exhibitedonly transient decreases in blood counts, although, importantly, thevalues did not fall below normal ranges.

We conclude that the anti-TROP-2 hRS7-CL2A-SN-38 ADC providedsignificant and specific antitumor effects against a range of humansolid tumor types. It was well tolerated in monkeys, with tissue TROP-2expression similar to humans. (Cardillo et al., 2011, Clin Cancer Res17:3157-69.)

Successful irinotecan treatment of patients with solid tumors has beenlimited due in large part to the low conversion rate of the CPT-11prodrug into the active SN-38 metabolite. Others have examinednontargeted forms of SN-38 as a means to bypass the need for thisconversion and to deliver SN-38 passively to tumors. We conjugated SN-38covalently to a humanized anti-TROP-2 antibody, hRS7. This antibody-drugconjugate has specific antitumor effects in a range of s.c. human cancerxenograft models, including non-small cell lung carcinoma, pancreatic,colorectal, and squamous cell lung carcinomas, all at nontoxic doses(e.g., ≤3.2 mg/kg cumulative SN-38 equivalent dose).

TROP-2 is widely expressed in many epithelial cancers, but also somenormal tissues, and therefore a dose escalation study in Cynomolgusmonkeys was performed to assess the clinical safety of this conjugate.Monkeys tolerated 24 mg SN-38 equivalents/kg with only minor,reversible, toxicities. Given its tumor-targeting and safety profile,hRS7-CL2A-SN-38 may provide an improvement in the management of solidtumors responsive to irinotecan.

Human trophoblast cell-surface antigen (TROP-2), also known as GA733-1(gastric antigen 733-1), EGP-1 (epithelial glycoprotein-1), and TACSTD2(tumor-associated calcium signal transducer), is expressed in a varietyof human carcinomas and has prognostic significance in some, beingassociated with more aggressive disease (see, e.g., Alberti et al.,1992, Hybridoma 11:539-45; Stein et al., 1993, Int J Cancer 55:938-46;Stein et al., 1994, Int J Cancer Suppl. 8:98-102). Studies of thefunctional role of TROP-2 in a mouse pancreatic cancer cell linetransfected with murine TROP-2 revealed increased proliferation in lowserum conditions, migration, and anchorage-independent growth in vitro,and enhanced growth rate with evidence of increased Ki-67 expression invivo and a higher likelihood to metastasize (Cubas et al., 2010, MolCancer 9:253).

TROP-2 antigen's distribution in many epithelial cancers makes it anattractive therapeutic target. Stein and colleagues (1993, Int J Cancer55:938-46) characterized an antibody, designated RS7-3G11 (RS7), thatbound to EGP-1, which was present in a number of solid tumors, but theantigen was also expressed in some normal tissues, usually in a lowerintensity, or in restricted regions. Targeting and therapeuticefficacies were documented in a number of human tumor xenografts usingradiolabeled RS7 (Shih et al., 1995, Cancer Res 55:5857s-63s; Stein etal., 1997, Cancer 80:2636-41; Govindan et al., 2004, Breast Cancer ResTreat 84:173-82), but this internalizing antibody did not showtherapeutic activity in unconjugated form (Shih et al., 1995, Cancer Res55:5857s-63s). However, in vitro it has demonstrated antibody-dependentcellular cytotoxicity (ADCC) activity against TROP-2 positivecarcinomas.

We reported the preparation of antibody-drug conjugates (ADC) using ananti-CEACAM5 (CD66e) IgG coupled to several derivatives of SN-38, atopoisomerase-I inhibitor that is the active component of irinotecan, orCPT-11 (Moon et al., 2008, J Med Chem 51:6916-26; Govindan et al., 2009,Clin Cancer Res 15:6052-61). The derivatives varied in their in vitroserum stability properties, and in vivo studies found one form(designated CL2) to be more effective in preventing or arresting thegrowth of human colonic and pancreatic cancer xenografts than otherlinkages with more or less stability.

Importantly, these effects occurred at nontoxic doses, with initialtesting failing to determine a dose-limiting toxicity (Govindan et al.,2009, Clin Cancer Res 15:6052-61). These results were encouraging, butalso surprising, because the CEACAM5 antibody does not internalize, aproperty thought to be critical to the success of an ADC. We speculatedthat the therapeutic activity of the anti-CEACAM5-SN-38 conjugate mightbe related to the slow release of SN-38 within the tumor after theantibody localized. Because irinotecan performs best when cells areexposed during the S-phase of their growth cycle, a sustained release isexpected to improve responses. Indeed, SN-38 coupled to nontargeting,plasma extending agents, such as polyethylene glycol (PEG) or micelles,has shown improved efficacy over irinotecan or SN-38 alone (e.g.,Koizumi et al., 2006, Cancer Res 66:10048-56), lending additionalsupport to this mechanism.

Given the RS7 antibody's broad reactivity with epithelial cancers andits internalization ability, we hypothesized that an RS7-SN-38 conjugatecould benefit not only from the sustained release of the drug, but alsofrom direct intracellular delivery. Therefore, we prepared and testedthe efficacy of SN-38 conjugates using a humanized version of the murineRS7 antibody (hRS7). A modification was made to the SN-38 derivative(Govindan et al., 2009, Clin Cancer Res 15:6052-61), which improved thequality of the conjugate without altering its in vitro stability or itsefficacy in vivo. This new derivative (designated CL2A) is currently thepreferred agent for SN-38 coupling to antibodies. Herein, we show theefficacy of the hRS7-SN-38 conjugate in several epithelial cancer celllines implanted in nude mice at nontoxic dosages, with other studiesrevealing that substantially higher doses could be tolerated. Moreimportantly, toxicity studies in monkeys that also express TROP-2 insimilar tissues as humans showed that hRS7-CL2A-SN-38 was tolerated atappreciably higher amounts than the therapeutically effective dose inmice, providing evidence that this conjugate is a promising agent fortreating patients with a wide range of epithelial cancers.

Materials and Methods

Cell Lines, Antibodies, and Chemotherapeutics. All human cancer celllines used in this study were purchased from the American Type CultureCollection. These include Calu-3 (non-small cell lung carcinoma),SK-MES-1 (squamous cell lung carcinoma), COLO 205 (colonicadenocarcinoma), Capan-1 and BxPC-3 (pancreatic adenocarcinomas), andPC-3 (prostatic adenocarcinomas). Humanized RS7 IgG and controlhumanized anti-CD20 (hA20 IgG, veltuzumab) and anti-CD22 (hLL2 IgG,epratuzumab) antibodies were prepared at Immunomedics, Inc. Irinotecan(20 mg/mL) was obtained from Hospira, Inc.

SN-38 Immunoconjugates and In Vitro Aspects. Synthesis of CL2-SN-38 hasbeen described previously (Moon et al., 2008, J Med Chem 51:6916-26, seealso U.S. Pat. No. 7,591,994). Its conjugation to hRS7 IgG and serumstability were performed as described (Moon et al., 2008, J Med Chem51:6916-26; Govindan et al., 2009, Clin Cancer Res 15:6052-61).Preparations of CL2A-SN-38 (M.W. 1480) and its hRS7 conjugate, andstability, binding, and cytotoxicity studies, were conducted asdescribed previously (Moon et al., 2008, J Med Chem 51:6916-26). Celllysates were prepared and immunoblotting for p21^(Waf1/Cip), p53, andPARP (poly-ADP-ribose polymerase) was performed.

In Vivo Therapeutic Studies. For all animal studies, the doses of SN-38immunoconjugates and irinotecan are shown in SN-38 equivalents. Based ona mean SN-38/IgG substitution ratio of 6, a dose of 500 μg ADC to a 20-gmouse (25 mg/kg) contains 0.4 mg/kg of SN-38. Irinotecan doses arelikewise shown as SN-38 equivalents (i.e., 40 mg irinotecan/kg isequivalent to 24 mg/kg of SN-38). NCr female athymic nude (nu/nu) mice,4 to 8 weeks old, and male Swiss-Webster mice, 10 weeks old, werepurchased from Taconic Farms. Tolerability studies were performed inCynomolgus monkeys (Macaca fascicularis; 2.5-4 kg male and female) bySNBL USA, Ltd. Animals were implanted subcutaneously with differenthuman cancer cell lines. Tumor volume (TV) was determined bymeasurements in 2 dimensions using calipers, with volumes defined as:L×w²/2, where L is the longest dimension of the tumor and w is theshortest. Tumors ranged in size between 0.10 and 0.47 cm³ when therapybegan. Treatment regimens, dosages, and number of animals in eachexperiment are described in the Results. The lyophilized hRS7-CL2A-SN-38and control ADC were reconstituted and diluted as required in sterilesaline. All reagents were administered intraperitoneally (0.1 mL),except irinotecan, which was administered intravenously. The dosingregimen was influenced by our prior investigations, where the ADC wasgiven every 4 days or twice weekly for varying lengths of time (Moon etal., 2008, J Med Chem 51:6916-26; Govindan et al., 2009, Clin Cancer Res15:6052-61). This dosing frequency reflected a consideration of theconjugate's serum half-life in vitro, to allow a more continuousexposure to the ADC.

Statistics. Growth curves were determined as percent change in initialTV over time. Statistical analysis of tumor growth was based on areaunder the curve (AUC). Profiles of individual tumor growth were obtainedthrough linear-curve modeling. An f-test was employed to determineequality of variance between groups before statistical analysis ofgrowth curves. A 2-tailed t-test was used to assess statisticalsignificance between the various treatment groups and controls, exceptfor the saline control, where a 1-tailed t-test was used (significanceat P≤0.05). Statistical comparisons of AUC were performed only up to thetime that the first animal within a group was euthanized due toprogression.

Pharmacokinetics and Biodistribution. ¹¹¹In-radiolabeled hRS7-CL2A-SN-38and hRS7 IgG were injected into nude mice bearing s.c. SK-MES-1 tumors(˜0.3 cm³). One group was injected intravenously with 20 μCi (250-μgprotein) of ¹¹¹In-hRS7-CL2A-SN-38, whereas another group received 20 μCi(250-μg protein) of ¹¹¹In-hRS7 IgG. At various timepoints mice (5 pertimepoint) were anesthetized, bled via intracardiac puncture, and theneuthanized. Tumors and various tissues were removed, weighed, andcounted by γ scintillation to determine the percentage injected dose pergram tissue (% ID/g). A third group was injected with 250 μg ofunlabeled hRS7-CL2A-SN-38 3 days before the administration of¹¹¹In-hRS7-CL2A-SN-38 and likewise necropsied. A 2-tailed t-test wasused to compare hRS7-CL2A-SN-38 and hRS7 IgG uptake after determiningequality of variance using the f-test. Pharmacokinetic analysis on bloodclearance was performed using WinNonLin software (Parsight Corp.).

Tolerability in Swiss-Webster Mice and Cynomolgus Monkeys. Briefly, micewere sorted into 4 groups each to receive 2-mL i.p. injections of eithera sodium acetate buffer control or 3 different doses of hRS7-CL2A-SN-38(4, 8, or 12 mg/kg of SN-38) on days 0 and 3 followed by blood and serumcollection, as described in Results. Cynomolgus monkeys (3 male and 3female; 2.5-4.0 kg) were administered 2 different doses ofhRS7-CL2A-SN-38. Dosages, times, and number of monkeys bled forevaluation of possible hematologic toxicities and serum chemistries aredescribed in the Results.

Results

Stability and Potency of hRS7-CL2A-SN-38. Two different linkages wereused to conjugate SN-38 to hRS7 IgG. The first is termed CL2-SN-38 andhas been described previously (Moon et al., 2008, J Med Chem 51:6916-26;Govindan et al., 2009, Clin Cancer Res 15:6052-61). A change was made tothe synthesis of the CL2 linker in that the phenylalanine moiety wasremoved. This change simplified the synthesis, but did not affect theconjugation outcome (e.g., both CL2-SN-38 and CL2A-SN-38 incorporated ˜6SN-38 per IgG molecule). Side-by-side comparisons found no significantdifferences in serum stability, antigen binding, or in vitrocytotoxicity (not shown).

To confirm that the change in the SN-38 linker from CL2 to CL2A did notimpact in vivo potency, hRS7-CL2A and hRS7-CL2-SN-38 were compared inmice bearing COLO 205 or Capan-1 tumors (not shown), using 0.4 mg or 0.2mg/kg SN-38 twice weekly×4 weeks, respectively, and with starting tumorsof 0.25 cm³ size in both studies. Both the hRS7-CL2A and CL2-SN-38conjugates significantly inhibited tumor growth compared to untreated(AUC_(14 days)P<0.002 vs. saline in COLO 205 model; AUC_(21 days)P<0.001vs. saline in Capan-1 model), and a nontargeting anti-CD20 control ADC,hA20-CL2A-SN-38 (AUC_(14 days)P<0.003 in COLO-205 model; AUC_(35 days):P<0.002 in Capan-1 model). At the end of the study (day 140) in theCapan-1 model, 50% of the mice treated with hRS7-CL2A-SN-38 and 40% ofthe hRS7-CL2-SN-38 mice were tumor-free, whereas only 20% of thehA20-ADC-treated animals had no visible sign of disease. Importantly,there were no differences in efficacy between the 2 specific conjugatesin both the tumor models.

Mechanism of Action. In vitro cytotoxicity studies demonstrated thathRS7-CL2A-SN-38 had IC₅₀ values in the nmol/L range against severaldifferent solid tumor lines (Table 9). The IC₅₀ with free SN-38 waslower than the conjugate in all cell lines. Although there was nocorrelation between TROP-2 expression and sensitivity tohRS7-CL2A-SN-38, the IC₅₀ ratio of the ADC versus free SN-38 was lowerin the higher TROP-2-expressing cells, most likely reflecting theenhanced ability to internalize the drug when more antigen is present.

TABLE 9 Expression of TROP-2 and in vitro cytotoxicity of SN-38 andhRS7-SN-38 in several solid tumor lines Cytotoxicity results TROP-2expression via FACS hRS7- Median SN-38 95% CI SN-38^(a) 95% CIfluorescence Percent IC₅₀ IC₅₀ IC₅₀ IC₅₀ ADC/free Cell line (background)positive (nmol/L) (nmol/L) (nmol/L) (nmol/L) SN-38 ratio Calu-3 282.2(4.7) 99.6% 7.19 5.77-8.95 9.97  8.12-12.25 1.39 COLO 141.5 (4.5) 99.5%1.02 0.66-1.57 1.95 1.26-3.01 1.91 205 Capan-1 100.0 (5.0) 94.2% 3.502.17-5.65 6.99 5.02-9.72 2.00 PC-3  46.2 (5.5) 73.6% 1.86 1.16-2.99 4.242.99-6.01 2.28 SK-  44.0 (3.5) 91.2% 8.61  6.30-11.76 23.14 17.98-29.782.69 MES-1 BxPC-3  26.4 (3.1) 98.3% 1.44 1.04-2.00 4.03 3.25-4.98 2.80^(a)IC₅₀-value is shown as SN-38 equivalents of hRS7-SN-38

SN-38 is known to activate several signaling pathways in cells, leadingto apoptosis. Our initial studies examined the expression of 2 proteinsinvolved in early signaling events (p21^(Waf1/Cip1) and p53) and 1 lateapoptotic event [cleavage of poly-ADP-ribose polymerase (PARP)] in vitro(not shown). In BxPC-3, SN-38 led to a 20-fold increase inp21^(waf1/Cip1) expression, whereas hRS7-CL2A-SN-38 resulted in only a10-fold increase, a finding consistent with the higher activity withfree SN-38 in this cell line (Table 9). However, hRS7-CL2A-SN-38increased p21^(Waf1/Cip1) expression in Calu-3 more than 2-fold overfree SN-38 (not shown).

A greater disparity between hRS7-CL2A-SN-38- and free SN-38-mediatedsignaling events was observed in p53 expression. In both BxPC-3 andCalu-3, upregulation of p53 with free SN-38 was not evident until 48hours, whereas hRS7-CL2A-SN-38 upregulated p53 within 24 hours (notshown). In addition, p53 expression in cells exposed to the ADC washigher in both cell lines compared to SN-38 (not shown). Interestingly,although hRS7 IgG had no appreciable effect on p21^(Waf1/Cip1)expression, it did induce the upregulation of p53 in both BxPC-3 andCalu-3, but only after a 48-hour exposure. In terms of later apoptoticevents, cleavage of PARP was evident in both cell lines when incubatedwith either SN-38 or the conjugate (not shown). The presence of thecleaved PARP was higher at 24 hours in BxPC-3, which correlates withhigh expression of p21 and its lower IC₅₀. The higher degree of cleavagewith free SN-38 over the ADC was consistent with the cytotoxicityfindings.

Efficacy of hRS7-SN-38. Because TROP-2 is widely expressed in severalhuman carcinomas, studies were performed in several different humancancer models, which started with an evaluation of the hRS7-CL2-SN-38linkage, but later, conjugates with the CL2A-linkage were used.Calu-3-bearing nude mice given 0.04 mg SN-38/kg of the hRS7-CL2-SN-38every 4 days×4 had a significantly improved response compared to animalsadministered the equivalent amount of hLL2-CL2-SN-38 (TV=0.14±0.22 cm³vs. 0.80±0.91 cm³, respectively; AUC_(42 days)P<0.026; FIG. 5A). Adose-response was observed when the dose was increased to 0.4 mg/kgSN-38. At this higher dose level, all mice given the specific hRS7conjugate were “cured” within 28 days, and remained tumor-free until theend of the study on day 147, whereas tumors regrew in animals treatedwith the irrelevant ADC (specific vs. irrelevant AUC_(98 days): P=0.05).In mice receiving the mixture of hRS7 IgG and SN-38, tumorsprogressed >4.5-fold by day 56 (TV=1.10±0.88 cm³; AUC_(56 days)P<0.006vs. hRS7-CL2-SN-38).

Efficacy also was examined in human colonic (COLO 205) and pancreatic(Capan-1) tumor xenografts. In COLO 205 tumor-bearing animals, (FIG.5B), hRS7-CL2-SN-38 (0.4 mg/kg, q4dx8) prevented tumor growth over the28-day treatment period with significantly smaller tumors compared tocontrol anti-CD20 ADC (hA20-CL2-SN-38), or hRS7 IgG (TV=0.16±0.09 cm³,1.19±0.59 cm³, and 1.77±0.93 cm³, respectively; AUC_(28 days)P<0.016).The MTD of irinotecan (24 mg SN-38/kg, q2dx5) was as effective ashRS7-CL2-SN-38, because mouse serum can more efficiently convertirinotecan to SN-38 than human serum, but the SN-38 dose in irinotecan(2,400 μg cumulative) was 37.5-fold greater than with the conjugate (64μg total).

Animals bearing Capan-1 showed no significant response to irinotecanalone when given at an SN-38-dose equivalent to the hRS7-CL2-SN-38conjugate (e.g., on day 35, average tumor size was 0.04±0.05 cm³ inanimals given 0.4 mg SN-38/kg hRS7-SN-38 vs. 1.78±0.62 cm³ inirinotecan-treated animals given 0.4 mg/kg SN-38; AUC_(day35)P<0.001;FIG. 5C). When the irinotecan dose was increased 10-fold to 4 mg/kgSN-38, the response improved, but still was not as significant as theconjugate at the 0.4 mg/kg SN-38 dose level (TV=0.17±0.18 cm³ vs.1.69±0.47 cm³, AUC_(day49)P<0.001). An equal dose of nontargetinghA20-CL2-SN-38 also had a significant antitumor effect as compared toirinotecan-treated animals, but the specific hRS7 conjugate wassignificantly better than the irrelevant ADC (TV=0.17±0.18 cm³ vs.0.80±0.68 cm³, AUC_(day49)P<0.018).

Studies with the hRS7-CL2A-SN-38 ADC were then extended to 2 othermodels of human epithelial cancers. In mice bearing BxPC-3 humanpancreatic tumors (FIG. 5D), hRS7-CL2A-SN-38 again significantlyinhibited tumor growth in comparison to control mice treated with salineor an equivalent amount of nontargeting hA20-CL2A-SN-38 (TV=0.24±0.11cm³ vs. 1.17±0.45 cm³ and 1.05±0.73 cm³, respectively;AUC_(day21)P<0.001), or irinotecan given at a 10-fold higher SN-38equivalent dose (TV=0.27±0.18 cm³ vs. 0.90±0.62 cm³, respectively;AUC_(day25)P<0.004). Interestingly, in mice bearing SK-MES-1 humansquamous cell lung tumors treated with 0.4 mg/kg of the ADC (FIG. 5E),tumor growth inhibition was superior to saline or unconjugated hRS7 IgG(TV=0.36±0.25 cm³ vs. 1.02±0.70 cm³ and 1.30±1.08 cm³, respectively;AUC_(28 days), P<0.043), but nontargeting hA20-CL2A-SN-38 or the MTD ofirinotecan provided the same antitumor effects as the specifichRS7-SN-38 conjugate. In all murine studies, the hRS7-SN-38 ADC was welltolerated in terms of body weight loss (not shown).

Biodistribution of hRS7-CL2A-SN-38. The biodistributions ofhRS7-CL2A-SN-38 or unconjugated hRS7 IgG were compared in mice bearingSK-MES-1 human squamous cell lung carcinoma xenografts (not shown),using the respective ¹¹¹In-labeled substrates. A pharmacokineticanalysis was performed to determine the clearance of hRS7-CL2A-SN-38relative to unconjugated hRS7 (not shown). The ADC cleared faster thanthe equivalent amount of unconjugated hRS7, with the ADC exhibiting ˜40%shorter half-life and mean residence time. Nonetheless, this had aminimal impact on tumor uptake (not shown). Although there weresignificant differences at the 24- and 48-hour timepoints, by 72 hours(peak uptake) the amounts of both agents in the tumor were similar.Among the normal tissues, hepatic and splenic differences were the moststriking (not shown). At 24 hours postinjection, there was >2-fold morehRS7-CL2A-SN-38 in the liver than hRS7 IgG. Conversely, in the spleenthere was 3-fold more parental hRS7 IgG present at peak uptake (48-hourtimepoint) than hRS7-CL2A-SN-38. Uptake and clearance in the rest of thetissues generally reflected differences in the blood concentration.

Because twice-weekly doses were given for therapy, tumor uptake in agroup of animals that first received a predose of 0.2 mg/kg (250 μgprotein) of the hRS7 ADC 3 days before the injection of the¹¹¹In-labeled antibody was examined. Tumor uptake of¹¹¹In-hRS7-CL2A-SN-38 in predosed mice was substantially reduced atevery timepoint in comparison to animals that did not receive thepredose (e.g., at 72 hours, predosed tumor uptake was 12.5%±3.8% ID/gvs. 25.4%±8.1% ID/g in animals not given the predose; P=0.0123).Predosing had no appreciable impact on blood clearance or tissue uptake(not shown). These studies suggest that in some tumor models, tumoraccretion of the specific antibody can be reduced by the precedingdose(s), which likely explains why the specificity of a therapeuticresponse could be diminished with increasing ADC doses and why furtherdose escalation is not indicated.

Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster Mice and CynomolgusMonkeys. Swiss-Webster mice tolerated 2 doses over 3 days, each of 4, 8,and 12 mg SN-38/kg of the hRS7-CL2A-SN-38, with minimal transient weightloss (not shown). No hematopoietic toxicity occurred and serumchemistries only revealed elevated aspartate transaminase (AST) andalanine transaminase (not shown). Seven days after treatment, AST roseabove normal levels (>298 U/L) in all 3 treatment groups (not shown),with the largest proportion of mice being in the 2×8 mg/kg group.However, by 15 days posttreatment, most animals were within the normalrange. ALT levels were also above the normal range (>77 U/L) within 7days of treatment (not shown) and with evidence of normalization by Day15. Livers from all these mice did not show histologic evidence oftissue damage (not shown). In terms of renal function, only glucose andchloride levels were somewhat elevated in the treated groups. At 2×8mg/kg, 5 of 7 mice had slightly elevated glucose levels (range of273-320 mg/dL, upper end of normal 263 mg/dL) that returned to normal by15 days postinjection. Similarly, chloride levels were slightlyelevated, ranging from 116 to 127 mmol/L (upper end of normal range 115mmol/L) in the 2 highest dosage groups (57% in the 2×8 mg/kg group and100% of the mice in the 2×12 mg/kg group), and remained elevated out to15 days postinjection. This also could be indicative of gastrointestinaltoxicity, because most chloride is obtained through absorption by thegut; however, at termination, there was no histologic evidence of tissuedamage in any organ system examined (not shown).

Because mice do not express TROP-2 bound by hRS7, a more suitable modelwas required to determine the potential of the hRS7 conjugate forclinical use. Immunohistology studies revealed binding in multipletissues in both humans and Cynomolgus monkeys (breast, eye,gastrointestinal tract, kidney, lung, ovary, fallopian tube, pancreas,parathyroid, prostate, salivary gland, skin, thymus, thyroid, tonsil,ureter, urinary bladder, and uterus; not shown). Based on thiscross-reactivity, a tolerability study was performed in monkeys.

The group receiving 2×0.96 mg SN-38/kg of hRS7-CL2A-SN-38 had nosignificant clinical events following the infusion and through thetermination of the study. Weight loss did not exceed 7.3% and returnedto acclimation weights by day 15. Transient decreases were noted in mostof the blood count data (not shown), but values did not fall belownormal ranges. No abnormal values were found in the serum chemistries.Histopathology of the animals necropsied on day 11 (8 days after lastinjection) showed microscopic changes in hematopoietic organs (thymus,mandibular and mesenteric lymph nodes, spleen, and bone marrow),gastrointestinal organs (stomach, duodenum, jejunum, ileum, cecum,colon, and rectum), female reproductive organs (ovary, uterus, andvagina), and at the injection site. These changes ranged from minimal tomoderate and were fully reversed at the end of the recovery period (day32) in all tissues, except in the thymus and gastrointestinal tract,which were trending towards full recovery at this later timepoint.

At the 2×1.92 mg SN-38/kg dose level of the conjugate, there was 1 deatharising from gastrointestinal complications and bone marrow suppression,and other animals within this group showed similar, but more severeadverse events than the 2×0.96 mg/kg group. These data indicate thatdose-limiting toxicities were identical to that of irinotecan; namely,intestinal and hematologic. Thus, the MTD for hRS7-CL2A-SN-38 liesbetween 2×0.96 and 1.92 mg SN-38/kg, which represents a human equivalentdose of 2×0.3 to 0.6 mg/kg SN-38.

Discussion

TROP-2 is a protein expressed on many epithelial tumors, including lung,breast, colorectal, pancreas, prostate, and ovarian cancers, making it apotentially important target for delivering cytotoxic agents. The RS7antibody internalizes when bound to TROP-2 (Shih et al., 1995, CancerRes 55:5857s-63s), which enables direct intracellular delivery ofcytotoxics.

Conjugation of chemotherapeutic drugs to antibodies has been exploredfor over 30 years. Because a substantial portion of an ADC is notprocessed by the tumor, but by normal tissues, there is a risk thatthese agents will be too toxic to normal organ systems before reachingthe therapeutic level in tumors. As with any therapeutic, thetherapeutic window is a key factor determining the potential of an ADC,and thus rather than examining “ultratoxic” drugs, we chose SN-38 as thedrug component of the TROP-2-targeted ADC.

SN-38 is a potent topoisomerase-I inhibitor, with IC₅₀ values in thenanomolar range in several cell lines. It is the active form of theprodrug, irinotecan, that is used for the treatment of colorectalcancer, and which also has activity in lung, breast, and brain cancers.We reasoned that a directly targeted SN-38, in the form of an ADC, wouldbe a significantly improved therapeutic over CPT-11, by overcoming thelatter's low and patient-variable bioconversion to active SN-38.

The Phe-Lys peptide inserted in the original CL2 derivative allowed forpossible cleavage via cathepsin B. In an effort to simplify thesynthetic process, in CL2A, phenylalanine was eliminated, and thus thecathepsin B cleavage site was removed. Interestingly, this product had abetter-defined chromatographic profile compared to the broad profileobtained with CL2 (not shown), but more importantly, this change had noimpact on the conjugate's binding, stability, or potency in side-by-sidetesting. These data suggest that SN-38 in CL2 was released from theconjugate primarily by the cleavage at the pH-sensitive benzyl carbonatebond to SN-38's lactone ring and not the cathepsin B cleavage site.

In vitro cytotoxicity of hRS7 ADC against a range of solid tumor celllines consistently had IC₅₀ values in the nmol/L range. However, cellsexposed to free SN-38 demonstrated a lower IC₅₀ value compared to theADC. This disparity between free and conjugated SN-38 was also reportedfor ENZ-2208 (Sapra et al., 2008, Clin Cancer Res 14:1888-96) and NK012(Koizumi et al., 2006, Cancer Res 66:10048-56). ENZ-2208 utilizes abranched PEG to link about 3.5 to 4 molecules of SN-38 per PEG, whereasNK012 is a micelle nanoparticle containing 20% SN-38 by weight. With ourADC, this disparity (i.e., ratio of potency with free vs. conjugatedSN-38) decreased as the TROP-2 expression levels increased in the tumorcells, suggesting an advantage to targeted delivery of the drug. Interms of in vitro serum stability, both the CL2- and CL2A-SN-38 forms ofhRS7-SN-38 yielded a t/_(1/2) of ˜20 hours, which is in contrast to theshort t/_(1/2) of 12.3 minutes reported for ENZ-2208 (Zhao et al., 2008,Bioconjug Chem 19:849-59), but similar to the 57% release of SN-38 fromNK012 under physiological conditions after 24 hours (Koizumi et al.,2006, Cancer Res 66:10048-56).

Treatment of tumor-bearing mice with hRS7-SN-38 (either with CL2-SN-38or CL2A-SN-38) significantly inhibited tumor growth in 5 different tumormodels. In 4 of them, tumor regressions were observed, and in the caseof Calu-3, all mice receiving the highest dose of hRS7-SN-38 weretumor-free at the conclusion of study. Unlike in humans, irinotecan isvery efficiently converted to SN-38 by a plasma esterase in mice, with agreater than 50% conversion rate, and yielding higher efficacy in micethan in humans. When irinotecan was administered at 10-fold higher orequivalent SN-38 levels, hRS7-SN-38 was significantly better incontrolling tumor growth. Only when irinotecan was administered at itsMTD of 24 mg/kg q2dx5 (37.5-fold more SN-38) did it equal theeffectiveness of hRS7-SN-38. In patients, we would expect this advantageto favor hRS7-CL2A-SN-38 even more, because the bioconversion ofirinotecan would be substantially lower.

We also showed in some antigen-expressing cell lines, such as SK-MES-1,that using an antigen-binding ADC does not guarantee better therapeuticresponses than a nonbinding, irrelevant conjugate. This is not anunusual or unexpected finding. Indeed, the nonbinding SN-38 conjugatesmentioned earlier enhance therapeutic activity when compared toirinotecan, and so an irrelevant IgG-SN-38 conjugate is expected to havesome activity. This is related to the fact that tumors have immature,leaky vessels that allow the passage of macromolecules better thannormal tissues. With our conjugate, 50% of the SN-38 will be released in˜13 hours when the pH is lowered to a level mimicking lysosomal levels(e.g., pH 5.3 at 37° C.; data not shown), whereas at the neutral pH ofserum, the release rate is reduced nearly 2-fold. If an irrelevantconjugate enters an acidic tumor microenvironment, it is expected torelease some SN-38 locally. Other factors, such as tumor physiology andinnate sensitivities to the drug, will also play a role in defining this“baseline” activity. However, a specific conjugate with a longerresidence time should have enhanced potency over this baseline responseas long as there is ample antigen to capture the specific antibody.Biodistribution studies in the SK-MES-1 model also showed that if tumorantigen becomes saturated as a consequence of successive dosing, tumoruptake of the specific conjugate is reduced, which yields therapeuticresults similar to that found with an irrelevant conjugate.

Although it is challenging to make direct comparisons between our ADCand the published reports of other SN-38 delivery agents, some generalobservations can be made. In our therapy studies, the highest individualdose was 0.4 mg/kg of SN-38. In the Calu-3 model, only 4 injections weregiven for a total cumulative dose of 1.6 mg/kg SN-38 or 32 μg SN-38 in a20 g mouse. Multiple studies with ENZ-2208 were done using its MTD of 10mg/kg×5, and preclinical studies with NK012 involved its MTD of 30mg/kg×3. Thus, significant antitumor effects were obtained withhRS7-SN-38 at 30-fold and 55-fold less SN-38 equivalents than thereported doses in ENZ-2208 and NK012, respectively. Even with 10-foldless hRS7 ADC (0.04 mg/kg), significant antitumor effects were observed,whereas lower doses of ENZ-2208 were not presented, and when the NK012dose was lowered 4-fold to 7.5 mg/kg, efficacy was lost (Koizumi et al.,2006, Cancer Res 66:10048-56). Normal mice showed no acute toxicity witha cumulative dose over 1 week of 24 mg/kg SN-38 (1,500 mg/kg of theconjugate), indicating that the MTD was higher. Thus, tumor-bearinganimals were effectively treated with 7.5- to 15-fold lower amounts ofSN-38 equivalents.

As a topoisomerase-I inhibitor, SN-38 induces significant damage to acell's DNA, with upregulation of p53 and p21^(WAF1/Cip1) resulting incaspase activation and cleavage of PARP. When we exposed BxPC-3 andCalu-3 cells to our ADC, both p53 and p21^(WAF1/Cip1) were upregulatedabove basal levels. In addition, PARP cleavage was also evident in bothcell lines, confirming an apoptotic event in these cells. Of interestwas the higher upregulation of p21^(Waf1/Cip1) in BxPC-3 and Calu-3relative to p53 by both free SN-38 and our hRS7-SN-38. This may beindicative of the mutational status of p53 in these 2 cell lines and theuse of a p53-independent pathway for p21^(Waf1/Cip1)-mediated apoptosis.

An interesting observation was the early upregulation of p53 in bothBxPC-3 and Calu-3 at 24 hours mediated by the hRS7-ADC relative to freeSN-38. Even the naked hRS7 IgG could upregulate p53 in these cell lines,although only after a 48-hour exposure. TROP-2 overexpression andcross-linking by antibodies has been linked to several MAPK-relatedsignaling events, as well as intracellular calcium release. Whilebinding of hRS7 was not sufficient to induce apoptosis in BxPC-3 andCalu-3, as evidenced by the lack of PARP cleavage, it may be enough toprime a cell, such that the inclusion of SN-38 conjugated to hRS7 maylead to a greater effect on tumor growth inhibition. Studies arecurrently underway to understand which pathways are involved withhRS7-delivery of SN-38 and how they may differ from free SN-38, and whateffect p53 status may play in this signaling.

Biodistribution studies revealed the hRS7-CL2A-SN-38 had similar tumoruptake as the parental hRS7 IgG, but cleared substantially faster with2-fold higher hepatic uptake, which may be due to the hydrophobicity ofSN-38. With the ADC being cleared through the liver, hepatic andgastrointestinal toxicities were expected to be dose limiting. Althoughmice had evidence of increased hepatic transaminases, gastrointestinaltoxicity was mild at best, with only transient loss in weight and noabnormalities noted upon histopathologic examination. Interestingly, nohematological toxicity was noted. However, monkeys showed an identicaltoxicity profile as expected for irinotecan, with gastrointestinal andhematological toxicity being dose-limiting.

Because TROP-2 recognized by hRS7 is not expressed in mice, it wascritically important to perform toxicity studies in monkeys that have asimilar tissue expression of TROP-2 as humans. Monkeys tolerated 0.96mg/kg/dose (˜12 mg/m²) with mild and reversible toxicity, whichextrapolates to a human dose of ˜0.3 mg/kg/dose (˜11 mg/m²). In a PhaseI clinical trial of NK012, patients with solid tumors tolerated 28 mg/m²of SN-38 every 3 weeks with Grade 4 neutropenia as dose-limitingtoxicity (Hamaguchi et al., 2010, Clin Cancer Res 16:5058-66).Similarly, Phase I clinical trials with ENZ-2208 revealed dose-limitingfebrile neutropenia, with a recommendation to administer 10 mg/m² every3 weeks or 16 mg/m² if patients were administered G-CSF. Because monkeystolerated a cumulative human equivalent dose of 22 mg/m², it is possiblethat even though hRS7 binds to a number of normal tissues, the MTD for asingle treatment of the hRS7 ADC could be similar to that of the othernontargeting SN-38 agents. Indeed, the specificity of the anti-TROP-2antibody did not appear to play a role in defining the DLT, because thetoxicity profile was similar to that of irinotecan. More importantly, ifantitumor activity can be achieved in humans as in mice that respondedwith human equivalent dose of just at 0.03 mg SN-38 equivalents/kg/dose,then significant antitumor responses could be realized clinically.

In conclusion, toxicology studies in monkeys, combined with in vivohuman cancer xenograft models in mice, have indicated that this ADCtargeting TROP-2 is an effective therapeutic in several tumors ofdifferent epithelial origin.

Example 13 Anti-CD22 (Epratuzumab) Conjugated-SN-38 for the Therapy ofHematologic Malgnancies

We previously found that slowly internalizing antibodies conjugated withSN-38 could be used successfully when prepared with a CL2A linker thatallows approximately 50% of the IgG-bound SN-38 to dissociate in serumevery 24 hours. In this study, the efficacy of SN-38 conjugates preparedwith epratuzumab (rapidly internalizing) and veltuzumab (slowlyinternalizing), humanized anti-CD22 and anti-CD20 IgG, respectively, wasexamined for the treatment of B-cell malignancies. Both antibody-drugconjugates had similar nanomolar activity against a variety of humanlymphoma/leukemia cell lines, but slow release of SN-38 compromisedpotency discrimination in vitro even against an irrelevant conjugate.When SN-38 was stably linked to the anti-CD22 conjugate, its potency wasreduced 40- to 55-fold. Therefore, further studies were conducted onlywith the less stable, slowly dissociating CL2A linker. In vivo, similarantitumor activity was found between CD22 and CD20 antibody-drugconjugate in mice-bearing Ramos xenografts, even though Ramos expressed15-fold more CD20 than CD22, suggesting that the internalization of theepratuzumab-SN-38 conjugate (Emab-SN-38) enhanced its activity.Emab-SN-38 was more efficacious than a nonbinding, irrelevant IgG-SN-38conjugate in vivo, eliminating a majority of well-established Ramosxenografts at nontoxic doses. In vitro and in vivo studies showed thatEmab-CL2A-SN-38 could be combined with unconjugated veltuzumab for amore effective treatment. Thus, Emab-SN-38 is active in lymphoma andleukemia at doses well below toxic levels and therefore represents a newpromising agent with therapeutic potential alone or combined withanti-CD20 antibody therapy. (Sharkey et al., 2011, Mol Cancer Ther11:224-34.)

Introduction

A significant effort has focused on the biologic therapy of leukemia andlymphoma, where unconjugated antibodies (e.g., rituximab, alemtuzumab,ofatumumab), radioimmunoconjugates (⁹⁰Y-ibritumomab tiuxetan,¹³¹I-tositumomab), and a drug conjugate (gemtuzumab ozogamicin) receivedU.S. Food and Drug Administration (FDA) approval. Another antibody-drugconjugate (ADC), brentuximab vedotin (SGN-35; anti-CD30-auristatin E),recently received accelerated approval by the FDA for Hodgkin lymphomaand anaplastic large-cell lymphomas. There are also a number of otherADCs in preclinical and clinical development that target CD19, CD22,CD37, CD74, and CD79b.

Antibodies against all of these targets are logical choices for carriersof drugs, because they are internalizing. Internalization andspecificity of CD22 have made it a particularly important target forleukemia and lymphomas, with at least 3 different anti-CD22 conjugatesin clinical investigation, including CMC-544 (acid-labile-conjugatedcalicheamicin), an anti-CD22-maytansine conjugate (stably linkedMCC-DM1), and CAT-3888 (formally BL22; a Pseudomonas exotoxinsingle-chain fusion protein). The active agent in all of theseconjugates has subnanomolar potency (i.e., so called ultra-toxics).

We recently developed methods to conjugate antibodies with SN-38, atopoisomerase I inhibitor with low nanomolar potency that is derivedfrom the prodrug, irinotecan (Govindan et al., 2009, Clin Cancer Res15:6052-62; Moon et al., 2008, J Med Chem 51:6916-26). Four SN-38linkage chemistries were examined initially using conjugates preparedwith a slowly internalizing anti-CEACAM5 antibody (Govindan et al.,2009, Clin Cancer Res 15:6052-62; Moon et al., 2008, J Med Chem51:6916-26). The conjugates retained CEACAM5 binding but differed in thedissociation rate of SN-38 in human serum, with half-lives varying fromapproximately 10 to 67 hours (Govindan et al., 2009, Clin Cancer Res15:6052-62). Ultimately, the linker designated CL2, with intermediatestability (˜50% dissociated in 24-35 hours), was selected for furtherdevelopment. CL2 was modified recently, eliminating the phenylalanine inthe cathepsin B-cleavable dipeptide to simplify and improvemanufacturing yields. The new derivative, designated CL2A, retains thepH-sensitive carbonate linkage to the SN-38, but it is no longerselectively cleaved by cathepsin B. Nevertheless, it has identical serumstability and in vivo activity as the original CL2 linker (Cardillo etal., 2011, Clin Cancer Res 17:3157-69). Because significant efficacywithout toxicity was found with the slowly internalizinganti-CEACAM5-SN-38, we postulated that its activity was aided by theslow release of SN-38 from the antibody after it localized in a tumor.Thus, the main objective in this report was to evaluate the therapeuticprospects of conjugates prepared using the CL2A linker with twoantibodies that are highly specific for B-cell cancers but differ intheir antigen expression and internalization properties.

Epratuzumab (Emab) is a rapidly internalizing (e.g., ≥50% within 1hour), humanized anti-CD22 IgG1 that has been evaluated extensively inlymphoma and leukemia in an unconjugated or conjugated form. Veltuzumab(Vmab) is a humanized anti-CD20 antibody that is also being studiedclinically but internalizes slowly (e.g., ˜10% in 1 hour). CD20 isusually expressed at much higher levels than CD22 in non-Hodgkinlymphoma, whereas CD22 is preferentially expressed in acutelymphoblastic leukemia (ALL) but not in multiple myeloma. Bothantibodies are effective in patients as unconjugated agents, but onlyveltuzumab is active in murine xenograft models (Stein et al., 2004,Clin Cancer Res 10:2868-76). On the basis of previous studies thatshowed ⁹⁰Y-Emab combined with unconjugated veltuzumab had enhancedefficacy in NHL models (Mattes et al., 2008, Clin Cancer Res14:6154-60), we also examined the Emab-SN-38+Vmab combination, as thiscould provide additional benefit without competing for the same targetantigen or having additional toxicity.

Materials and Methods

Cell Lines. Ramos, Raji, Daudi (Burkitt lymphomas), and JeKo-1 (mantlecell lymphoma) were purchased from American Type Culture Collection.REH, RS4; 11, MN-60, and 697 (ALL) were purchased from Deutsche Sammlungvon Mikroorganismen and Zellkulturen. WSU-FSCCL (follicular NHL) was thegift of Dr. Mitchell R. Smith (Fox Chase Cancer Center, Philadelphia,Pa.). All cell lines were cultured in a humidified CO₂ incubator (5%) at37° C. in recommended supplemented media containing 10 to 20% fetal calfserum and were checked periodically for Mycoplasma.

Antibodies and Conjugation Methods. Epratuzumab and veltuzumab arehumanized anti-CD22 and anti-CD20 IgG1 monoclonal antibodies,respectively. Labetuzumab (Lmab), a humanized anti-CEACAM5 IgG1, andRS7, a humanized anti-TROP-2 antibody (both from Immunomedics, Inc.),were used as nonbinding, irrelevant controls. Herein, Emab-SN-38,Vmab-SN-38, and Lmab-SN-38 refer to conjugates prepared using the CL2Alinker that was described above. In vitro studies in human serum showedthat approximately 50% of the active SN-38 moiety is released from theIgG each day (Cardillo et al., 2011, Clin Cancer Res 17:3157-69).Another linker, designated CL2E (see U.S. Pat. Nos. 7,999,083 and8,080,250), is stable in human serum over 14 days, but it contains acathepsin B cleavage site to facilitate the release of SN-38 whenprocessed in lysosomes. The method to prepare CL2E and the structures ofthe CL2A and CL2E linkers are given in t U.S. Pat. Nos. 7,999,083 and8,080,250. The conjugates contained approximately 6 SN-38 units per IgG(e.g., 1.0 mg of the IgG-SN-38 conjugate contains ˜16 μg of SN-38).

In Vitro Cell Binding and Cytotoxicity. Flow cytometry was carried outusing the unconjugated specific and irrelevant antibodies incubated for1 hour at 4° C., with binding revealed using fluorescein isothiocyanate(FITC)-Fcγ fragment-specific goat anti-human IgG (JacksonImmunoResearch), also incubated for 1 hour at 4° C. Median fluorescencewas determined on a FACSCALIBUR® flow cytometer (Becton Dickinson) usinga CellQuest software package.

Cytotoxicity was determined using the MTS dye reduction assay (Promega).Dose-response curves [with/without goat anti-human Fcγ F(ab′)₂; JacksonImmunoResearch] were generated from the mean of triplicatedeterminations, and IC₅₀-values were calculated using PRISM® GraphPadsoftware (v5), with statistical comparisons using an F test on the bestfit curves for the data. Significance was set at P<0.05.

Immunoblotting. After 24- or 48-hour exposure to the test agents,markers of early (p21 expression) and late (PARP cleavage) apoptosiswere revealed by Western blotting.

In Vivo Studies. The subcutaneous Ramos model was initiated byimplanting 1×10⁷ cells (0.2 mL) from culture (>95% viability) into 4- to6-week-old female nude mice (Taconic). Three weeks from implantation,animals with tumors ranging from 0.4 to 0.8 cm³ (measured by caliper,L×W×D) were segregated into groups of animals, each with the same rangeof tumor sizes. Tumor size and body weights were measured at least onceweekly, with animals removed from the study when tumors grew to 3.0 cm³or if they experienced 20% or greater body weight loss. The intravenousWSU-FSCCL and 697 models were initiated by intravenous injection of2.5×10⁶ and 1×10⁷ cells, respectively, in female severe combinedimmunodeficient (SCID) mice (Taconic). Treatment began 5 days afteradministration of the WSU-FSCCL cells and 7 days after the 697inoculation. Animals were observed daily, using hind leg paralysis orother signs of morbidity as surrogate survival endpoints. All treatmentswere given intraperitoneally in ≤0.2 mL. The specific dosages andfrequency are given in the Results section. Because mice convertirinotecan to SN-38 efficiently, irinotecan dosing was adjusted on thebasis of SN-38 equivalents; SN-38 mole equivalents are based on 1.6% ofADC mass and 60% of irinotecan mass.

Efficacy was expressed in a Kaplan-Meier curve, using time toprogression (TTP) as surrogate survival endpoints as indicated above.Statistical analysis was conducted by a log-rank test using PRISM®GraphPad software (significance, P<0.05).

Results

Antigen Expression and Cytotoxicity In Vitro. All cell lines were highlysusceptible to SN-38, with EC₅₀ values ranging from 0.13 nmol/L forDaudi to 2.28 nmol/L for RS4; 11 (Table 10). Except for 697 and RS4; 11,the Emab-SN-38 anti-CD22 conjugate was 2- to 7-fold less effective thanSN-38. This is a common finding with our targeted, as well as othernontargeted, SN-38 conjugates. Despite differences in antigenexpression, the Emab-CL2A-SN-38 and Vmab-CL2A-SN-38 had similarpotencies as the nonbinding, Lmab-CL2A-SN-38 anti-CEACAM5 conjugate,which was likely due to dissociation of approximately 90% of SN-38during the 4-day MTS assay. Other in vitro procedures using shorterexposure times were also ineffective in discriminating differences inthe potencies of conjugates. For example, Annexin V staining after a1-day exposure failed to find differences between untreated and treatedcells (not shown). Upregulation of p21 and PARP cleavage was alsoexamined as early and late markers of apoptosis, respectively. Ramos didnot express p21. However, PARP cleavage was detected, but only after a48-hour exposure, being more strongly expressed in SN-38-treated cells(not shown). The WSU-FSCCL cell line expressed p21, but neither p21upregulation nor PARP cleavage was evident until 48 hours afterEmab-CL2A-SN-38 exposure. However, both were observed after a 24-hourexposure with free SN-38 (not shown). While the enhanced intensity andearlier activation of apoptotic events with free SN-38 are consistentwith its lower EC₅₀ over the IgG-conjugated form, the results indicatedthat an exposure period of at least 48 hours would be required, but atthis time, approximately 75% of the SN-38 would be released from theconjugate.

TABLE 10 Expression of CD20 and CD22 by FACScan and in vitrocytotoxicity by MTS assay of SN-38 and specific Emab anti-CD22-SN-38,Vmab anti-CD20-SN-38, and Lmab anti- CEACAM5-SN-38 conjugates againstseveral hematopoietic tumor cell lines CD20 expression CD22 expressionEC₅₀ values^(a) Median Median Emab- Vmab- Lmab- fluorescencefluorescence SN-38, 95% SN-38, 95% SN-38, 95% SN-38, 95% Cell line(background) (background) nmol/L CI nmol/L CI nmol/L CI nmol/L CINHL:Burkitt Raji 422.2 (6.8) 45.9 (6.8) 1.42 0.8-2.4 2.10 1.2-3.8 ND —ND — 4.61 2.2-9.5 4.88 2.7-9.0 3.73 1.8-7.6 Ramos 620.4 (4.1) 40.8 (4.1)0.40 0.2-0.7 2.92 1.6-5.4 ND — ND — 9.84  4.5-21.6 13.56   4.9-37.2 8.08 2.9-22.2 Daudi 815.1 (5.9) 145.0 (5.9)  0.13 0.1-0.2 0.52 0.4-0.7 ND —ND — NHL:follicular WSU-  97.4 (4.9)  7.7 (4.9) 0.50 0.3-1.0 0.680.4-1.1 ND — ND — FSCCL 1.05 0.8-1.4 0.83 0.6-1.1 1.17 0.8-1.7NHL:mantle cell Jeko-1 604.6 (6.5) 11.2 (6.5) ND — 2.25 1.3-3.8 1.981.1-3.5 2.27 1.3-3.9 ALL:B cell REH  12.3 (4.1) 22.9 (4.1) 0.47 0.3-0.91.22 0.8-1.9 ND — ND — 697  6.9 (4.2) 16.0 (4.2) 2.23 1.3-3.9 2.671.7-3.7 ND — ND — RS4; 11  3.7 (4.1) 23.3 (4.1) 2.28 1.1-4.9 1.681.0-3.0 ND — ND — MN-60  21.5 (5.8) 10.3 (5.8) 1.23 0.6-2.1 3.65 2.2-6.2ND — ND — Abbreviations: CI, confidence interval; ND, not determined.^(a)EC₅₀ expressed as mole equivalents of SN-38 in Emab-SN-38.

We again examined PARP cleavage and p21 expression, this time in cellstreated with Emab-CL2A-SN-38+Vmab. Confirming the earlier study inRamos, PARP cleavage first occurs only after a 48-hour exposure to theconjugate, with expression unchanged in the presence of a cross-linkingantibody (not shown). Exposure to veltuzumab for more than 48 hours hadno effect on PARP cleavage, but cleavage was strong within 24 hours whena cross-linking antibody was added (not shown). However, when veltuzumabalone (no cross-linker) was combined with Emab-CL2A-SN-38, PARP cleavageoccurred after a 24-hour exposure (not shown), indicating veltuzumabcould induce a more rapid onset of apoptosis, even in the absence ofcross-linking. The only notable difference in the WSU-FSCCL cell linewas that the combination greatly enhanced p21 expression at 48 hours(not shown), again suggesting an acceleration of apoptosis inductionwhen veltuzumab is combined with the Emab-CL2A-SN-38 conjugate. Thedelay in apoptosis induction in WSU-FSCCL as compared with Ramos islikely explained by the lower expression of CD22 and CD20.

Ultratoxic agents often use linkers that are highly stable in serum, astheir premature release would increase toxicity, but these conjugatesmust be internalized for the drug to be delivered optimally. Becauseepratuzumab internalizes rapidly, we examined whether it might benefitfrom a more stably linked SN-38, comparing in vitro cytotoxicity of theCL2A-linked Emab-SN-38 conjugate with the serum-stable CL2E-SN-38conjugate. Both conjugates had a similar binding affinity (not shown),but the more stable Emab-CL2E-SN-38 was approximately 40- to 55-timesless potent than the CL2A conjugate in 3 cell lines (not shown). Whilespecificity was lacking with the CL2A conjugates, the Emab-CL2E-SN-38consistently was approximately two times more potent than the nonbindingLmab-anti-CEACAM5-CL2E-SN-38 conjugate (not shown). We concluded that itwas unlikely that the more stably linked conjugate would be appropriatefor a slowly internalizing veltuzumab conjugate and therefore continuedour investigation only with CL2A-linked SN-38 conjugates.

Because of limitations of the in vitro assays, efficacy was assessed inxenograft models. As indicated in Table 10, all of the lymphoma celllines have much higher expression of CD20 than CD22. Daudi had thehighest expression of CD22 and CD20, but it is very sensitive in vivo tounconjugated veltuzumab and in vitro testing revealed the highestsensitivity to SN-38 (Table 10). These properties would likely make itdifficult to assess differences in activity attributed to the SN-38conjugate versus the unconjugated antibody, particularly whenunconjugated epratuzumab is not an effective therapeutic in animals.Because Ramos had been used previously to show an advantage forcombining ⁹⁰Y-Emab with veltuzumab (Mattes et al., 2008, Clin Cancer Res14:6154-60), we elected to start with a comparison of theEmab-CL2A-SN-38 and Vmab-CL2A-SN-38 conjugates in the Ramos humanBurkitt cell line. Despite flow cytometry showing a 15-fold higherexpression of CD20 over CD22, immunohistology of Ramos xenografts showedabundant CD22 and CD20, with CD22 seemingly expressed more uniformlythan CD20 (not shown).

Ramos xenografts in untreated animals progressed rapidly, reaching the3.0-cm³ termination size from their starting size of 0.4 cm³ within 6days (not shown), and as reported previously, neither veltuzumab norepratuzumab appreciably affected the progression of well-establishedRamos xenografts (Sharkey et al., 2009, J Nucl Med 50:444-53).Consistent with previous findings using other SN-38 conjugates, none ofthe animals treated with a 4-week, twice-weekly, 0.5 mg/dose treatmentregimen had appreciable weight loss. Both conjugates were highlyeffective in controlling tumor growth, with 80% or more of the animalshaving no evidence of tumor by the end of the 4-week treatment (FIG. 6).The 0.25-mg Vmab-CL2A-SN-38 dose was better at controlling growth overthe first 4 weeks, but at 0.5 mg, similar early growth control wasobserved for both conjugates. Thus, despite a 15-fold higher expressionof CD20 than CD22, Emab-CL2A-SN-38 compared favorably withVmab-CL2A-SN-38. Therefore, the remaining studies focused on Emab-SCL2A-N-38 alone or in combination with unconjugated veltuzumab.

Emab-CL2A-SN-38 dose-response and specificity. A dose-responserelationship was seen for the specific Emab-CL2A-SN-38 and irrelevantLmab-CL2A-SN-38 conjugates, but Emab-CL2A-SN-38 had significantly bettergrowth control at 2 of the 3 levels tested, and with a strong trendfavoring the specific conjugate at the intermediate dose (FIG. 7).Again, 0.25 mg of Emab-CL2A-SN-38 ablated a majority of the tumors;here, 7 of 10 animals were tumor-free at the end of the 12-weekmonitoring period, with no change in body weight. Animals givenirinotecan alone (6.5 μg/dose; approximately the same SN-38 equivalentsas 0.25 mg of conjugate) had a median survival of 1.9 weeks, with 3 of11 animals tumor-free at the end of the study, which was notsignificantly different from the 3.45-week median survival for theirrelevant Lmab-CL2A-SN-38 conjugate (P=0.452; FIG. 7C).

In the 697-disseminated leukemia model, the median survival ofsaline-treated animals was just 17 days from tumor inoculation. Animalsgiven unconjugated epratuzumab plus irinotecan (same mole equivalents ofSN-38 as 0.5 mg of the conjugate) had the same median survival, whereasanimals given 0.5 mg of Emab-CL2A-SN-38 twice weekly starting 7 daysfrom tumor inoculation survived to 24.5 days, significantly longer thanuntreated animals (P<0.0001) or for unconjugated epratuzumab given withirinotecan (P=0.016). However, Emab-CL2A-SN-38 was not significantlybetter than the irrelevant conjugate (median survival=22 days; P=0.304),most likely reflecting the low expression of CD22 in this cell line.

Emab-CL2A-SN-38 Combined with Unconjugated Vmab Anti-CD20. We previouslyreported improved responses when ⁹⁰Y-Emab was combined with unconjugatedveltuzumab in the subcutaneous Ramos model (Mattes et al., 2008, ClinCancer Res 14:6154-60) and thus this possibility was examined withEmab-CL2A-SN-38. In a pilot study, 5 animals bearing subcutaneous Ramostumors averaging approximately 0.3 cm³ were given veltuzumab (0.1 mg),0.1 mg of Emab-CL2A-SN-38, or Emab-CL2A-SN-38+Vmab (all agents giventwice weekly for 4 weeks). The median TTP to 2.0 cm³ was 22, 14, andmore than 77 days, respectively (veltuzumab vs. Emab-CL2A-SN-38 alone,P=0.59; Emab-CL2A-SN-38+Vmab vs. Emab-CL2A-SN-38, P=0.0145), providingan initial indication that the combination of veltuzumab withEmab-CL2A-SN-38 improved the overall therapeutic response. In afollow-up study that also used a twice-weekly, 4-week treatment regimen,6 of 11 animals given 0.1 mg of Emab-CL2A-SN-38 plus 0.1 mg ofveltuzumab had no evidence of tumors 16 weeks from the start oftreatment, whereas the median survival for animals receiving veltuzumabalone or with 0.1 mg of the control Lmab-CL2A-SN-38 was 1.9 and 3.3weeks, respectively, with 3 of 11 animals being tumor-free at 16 weeksin each of these groups (not shown). Despite the longer median TTP andmore survivors, no significant differences were found between thegroups. Thus, in the Ramos model, which has abundant CD20 and moderatelevels of CD22, the Emab-CL2A-SN-38 conjugate given at nontoxic doselevels was not significantly better than unconjugated anti-CD20 therapy,but the addition of Emab-CL2A-SN-38 to unconjugated anti-CD20 therapyappeared to improve the response without toxicity. It is important toemphasize that the SN-38 conjugates are given at levels far less thantheir maximum tolerated dose, and therefore these results should not beinterpreted that the unconjugated anti-CD20 therapy is equal to that ofthe Emab-CL2A-SN-38 conjugate.

Two additional studies were conducted in an intravenous implanted modelusing the WSU-FSCCL follicular NHL cell line that has a low expressionof CD20 and CD22 (not shown). The median survival time forsaline-treated animals was 40 to 42 days from tumor implantation.Irinotecan alone (not shown), given at a dose containing the same SN-38equivalents as 0.3 mg of the ADC, increased the median survival (49 vs.40 days, respectively; P=0.042), but 14 of 15 animals succumbed todisease progression on day 49, the same day the final 4 of 15 animals inthe saline group were eliminated (not shown). Despite its relatively lowCD20 expression, veltuzumab alone (35 μg twice weekly×4 weeks) waseffective in this model. The median survival increased to 91 days in thefirst study, with 2 cures (day 161), and to 77 days in the second, butwith no survivors after 89 days (veltuzumab alone vs. saline-treated,P<0.001 in both studies). Unconjugated epratuzumab (0.3 mg/dose)combined with irinotecan and veltuzumab had the same median survival asveltuzumab alone, suggesting that neither epratuzumab nor irinotecancontributed to the net response.

As expected because of the low CD22 expression by WSU-FSCCL,Emab-CL2A-SN-38 alone was not as effective as in Ramos. At the 0.15-mgdose, no significant benefit over the saline group was seen, but at 0.3mg, the median survival increased to 63 days, providing a significantimprovement compared with the saline-treated animals (P=0.006). Thesecond study, using 0.3 mg of Emab-CL2A-SN-38, confirmed an enhancedsurvival compared with the saline group (75 vs. 40 days; P<0.0001). Thespecificity of this response was not apparent in the first study, wherethe median survival of the irrelevant Lmab-CL2A-SN-38 conjugate andEmab-CL2A-SN-38 were not different at either 0.15- or 0.3-mg dose levels(42 vs. 49 days and 63 vs. 63 days for the Emab-CL2A-SN-38 vs.anti-CEACAM5CL2A-SN-38 conjugates at the 2 doses levels, respectively).However, in the second study, the 0.3-mg dose of Emab-CL2A-SN-38provided a significantly improved survival over the irrelevant conjugate(75 vs. 49 days; P<0.0001). Again, the difficulty in showing specificityin this model is most likely related to low CD22 expression.

Combining the specific Emab-CL2A-SN-38 with veltuzumab substantiallyincreases survival, with evidence of more robust responses than thecontrol Lmab-CL2A-SN-38. For example, in the first study, animalstreated with veltuzumab plus 0.15 or 0.3 mg of the control conjugate hada median survival of 98 and 91 days, respectively, which was similar tothat of veltuzumab alone (91 days; not shown). However, veltuzumab plus0.15 mg of the specific Emab-CL2A-SN-38 conjugate increased the mediansurvival to 140 days. While this improvement was not significantlyhigher than veltuzumab alone (P=0.257), when the Emab-CL2A-SN-38 dosewas increased to 0.3 mg with veltuzumab, 6 of 10 animals remained aliveat the end of the study, providing a significant survival advantage overthe control conjugate plus veltuzumab (P=0.0002). In a second study, themedian survival of veltuzumab alone was shorter than in the first (77vs. 91 days), yet the median survival for the control conjugate withveltuzumab was again 91 days, which now yielded a significant survivaladvantage over veltuzumab alone (P<0.0001). Combining the specificEmab-CL2A-SN-38 conjugate with veltuzumab extended the median survivalto 126 days, which was significantly longer than the median survival of75 and 77 days for Emab-CL2A-SN-38 and veltuzumab alone, respectively(P<0.0001 for each). However, in this study, it did not quite meet therequirements for a statistical improvement over the combination withcontrol anti-CEACAM5CL2A-SN-38 conjugate (P=0.078).

Discussion

Over the past 10 years, ADCs have made substantial gains in cancertherapy, yet there also have been some setbacks. The gains occurredlargely when investigators chose to examine agents that were too toxicto be used alone, but when coupled to an antibody, these so-calledultratoxics produced substantially improved responses in preclinicaltesting. The recent approval of brentuximab vedotin, an auristatinconjugate, in Hodgkin lymphoma and the clinical success withtrastuzumab-DM1 anti-HER2-maytansine conjugate as a single agent inbreast cancer refractive to unconjugated trastuzumab suggest that theseADCs bearing ultratoxic agents are becoming accepted treatmentmodalities. However, conjugates prepared with agents that are themselvespotent in the picomolar range can have an increased risk for toxicity,as the recent decision to withdraw gemtuzumab ozogamicin, theanti-CD33calicheamicin conjugate, from the market suggests (Ravandi,2011, J Clin Oncol 29:349-51). Thus, the success of an ADC may depend onidentifying appropriate chemistries to bind the drug and antibodytogether, as well as defining a suitable target that is sufficientlyexpressed to allow an adequate and selective delivery of the cytotoxicagent.

We developed a CL2A linker for coupling SN-38 to IgG that allows SN-38to be released slowly from the conjugate in serum (about 50% per day).With this linker, an antibody that is slowly internalized could be aneffective therapeutic, perhaps because the conjugate localized to atumor releases a sufficient amount of drug locally, even without beinginternalized. The CL2A linker also was used recently with an antibody toTROP-2 that was reported to be internalized rapidly (Cardillo et al.,2011, Clin Cancer Res 17:3157-69.). Thus, it appears that the slowrelease mechanism is beneficial for internalizing and non-internalizingantibodies.

In this report, we expanded our assessment of the CL2A linker bycomparing SN-38 conjugates prepared with epratuzumab, a rapidlyinternalizing anti-CD22 IgG, and veltuzumab, a slowly internalizinganti-CD20 IgG, for the treatment of B-cell malignancies. Prior studieswith the murine parent of epratuzumab had indicated that most of theantibody internalizes within 1 hour and 50% of CD22 is reexpressed onthe cell surface within 5 hours (Shih et al., 1994, Int J Cancer56:538-45). This internalization and reexpression process would permitintracellular delivery that might compensate for lower surfaceexpression of CD22. Because many of the B-cell malignancies express muchmore CD20 than CD22, a conjugate targeting CD20 might deliver more molesof drug by releasing its toxic payload after being localized in thetumor.

In vitro cytotoxicity studies could not discriminate the potency of thespecific conjugates or even an irrelevant conjugate because of therelease of SN-38 from the conjugate into the media. Indeed, SN-38 alonewas somewhat more potent than the conjugates, which may reflect itsaccelerated ability to enter the cell and engage topoisomerase I.Because other studies revealed that the conjugates required a 48-hourexposure before early signs of apoptosis could be seen, we concludedthat in vitro testing would not be able to discriminate the potency ofthese 2 conjugates and therefore resorted to in vivo studies.

In xenograft models, both conjugates had similar antitumor activityagainst Ramos tumors, which flow cytometry had indicated expressednearly 15-fold more CD20 than CD22. This lent support to selecting theEmab anti-CD22-CL2A-SN-38 conjugate especially because it could becombined with unconjugated Vmab anti-CD20 therapy without concern thateither agent would interfere with the binding of the other agent.Indeed, if an anti-CD20-CL2A-SN-38 conjugate were used, the total IgGprotein dose given likely would be below a level typically needed foreffective unconjugated anti-CD20 antibody treatments, as thedose-limiting toxicity would be driven by the SN-38 content. Adding moreunlabeled anti-CD20 to an anti-CD20-CL2A-SN-38 conjugate would riskreducing the conjugate's uptake and potentially diminishing itsefficacy. However, as we showed previously in combination studies usingradiolabeled epratuzumab with unconjugated veltuzumab, benefit can bederived from both agents given at their maximum effective and safedosages. In vitro studies showed veltuzumab, even in the absence ofcross-linking that is used to enhance signaling, accelerated apoptoticevents initiated with Emab-CL2A-SN-38. Thus, as long as theEmab-CL2A-SN-38 conjugate was as effective as the anti-CD20 conjugate,selecting the Emab-CL2A-SN-38 conjugate is a logical choice because itallows for a more effective combination therapy, even in tumors whereone or both of the antigens are low in expression.

Because most ADCs using ultratoxic drugs are stably linked, we alsotested a serum-stable, but intracellularly cleavable,anti-CD22CL2E-SN-38 conjugate, but determined it was 40- to 55-fold lesspotent than with the CL2A linker. Others have examined a variety ofultratoxic drugs conjugated to anti-CD20 or anti-CD22 antibodies,finding that internalizing conjugates are generally more active, butalso observing that even slowly internalizing antibodies could beeffective if the released drug penetrated the cell membrane. While theCL2A-type linker may be appropriate for SN-38, it may not be optimal fora more toxic agent, where even a small, sustained release in the serumwould increase toxicity and compromise the therapeutic window.

Emab-CL2A-SN-38 was active at a cumulative dose of 0.6 mg in micebearing Ramos (75 μg twice weekly for 4 weeks), which extrapolates to ahuman dose of just 2.5 mg/kg. Thus, Emab-CL2A-SN-38 should have an ampletherapeutic window in patients. Furthermore, an effective and safe doseof the anti-TROP-2CL2A-SN-38 conjugate was combined with a maximumtolerated dose of a ⁹⁰Y-labeled antibody without an appreciable increasein toxicity but with improved efficacy (Sharkey et al., 2011, Mol CancerTher 10:1072-81). Thus, the safety and efficacy profile of these SN-38antibody conjugates are very favorable for other combination therapies.

Even though irinotecan is not used routinely for the treatment ofhematopoietic cancers, SN-38 was as potent in lymphoma and leukemia celllines as in solid tumors (Cardillo et al., 2011, Clin Cancer Res17:3157-69.). In the WSU-FSCCL cell line, the specific and irrelevantIgG conjugates were significantly better than irinotecan, whereas inRamos, the median TTP with the irrelevant conjugate was longer but notsignificantly better than irinotecan. These results are consistent withother studies that have shown that a nonspecific IgG is an excellentcarrier for drugs and more potent in vivo than free drug or conjugatesprepared with albumin or polyethylene glycol (PEG)-Fc. While thePEG-SN-38 conjugate had significant antitumor effects, it was given atits maximum tolerated amounts, ranging from 10 to 30 mg/kg SN-38equivalents (Sapra et al., 2009, Haematologica 94:1456-9). In contrast,the maximum cumulative dose of SN-38 given over 4 weeks to animalsbearing Ramos was only 1.6 mg/kg (i.e., dosing of 0.25 mg of Emab-SN-38given twice weekly over 4 weeks) and this was nontoxic.

The specific therapeutic activity of Emab-CL2A-SN-38 appeared to improvein cell lines with higher CD22 expression. For example, in Ramos,specific therapeutic effects of Emab-CL2A-SN-38 alone were recorded at 2of the 3 different dose levels examined, and a sizeable number of tumorswere completely ablated. In contrast, in WSU-FSCCL that had about2.5-fold lower expression of CD22, Emab-CL2A-SN-38 improved survivalsignificantly compared with the irrelevant anti-CEACAM5-CL2A-SN-38conjugate in 1 of 2 studies. However, it is important to emphasize thatwhen used in combination with unconjugated anti-CD20 therapy,Emab-CL2A-SN-38 amplifies the therapeutic response. Thus, thecombination of these two treatments could augment the response even insituations where CD22 is not highly expressed.

In conclusion, using the less-stable CL2A-SN-28 linker, Emabanti-CD22-CL2A-SN-38 conjugate was equally active at nontoxic doses invivo as a similar anti-CD20-CL2A-SN-38 conjugate, despite the fact thatCD20 expression was more than a log-fold higher than CD22. Therapeuticresponses benefited by the combination of Emab-CL2A-SN-38 withunconjugated Vmab anti-CD20 therapy, even when CD22 expression was low,suggesting that the combination therapy could improve responses in anumber of B-cell malignancies when both antigens are present The currentstudies suggest that this combination is very potent in diverse lymphomaand leukemia preclinical models, yet appears to have less host toxicity.

Example 14 Anti-CD74-CL2A-SN-38 Conjugates for Treatment of CD74+HumanCancers

CD74 is an attractive target for antibody-drug conjugates (ADC), becauseit internalizes and recycles after antibody binding. CD74 mostly isassociated with hematological cancers, but is expressed also in solidcancers. Therefore, the utility of ADCs prepared with the humanizedanti-CD74 antibody, milatuzumab, for the therapy CD74-expressing solidtumors was examined. Milatuzumab-doxorubicin and two milatuzumab-SN-38conjugates were prepared with cleavable linkers (CL2A and CL2E),differing in their stability in serum and how they release SN-38 in thelysosome. CD74 expression was determined by flow cytometry andimmunohistology. In vitro cytotoxicity and in vivo therapeutic studieswere performed in the human cancer cell lines A-375 (melanoma), HuH-7and Hep-G2 (hepatoma), Capan-1 (pancreatic), and NCI-N87 (gastric), andRaji Burkitt lymphoma. The milatuzumab-SN-38 ADC was compared to SN-38ADCs prepared with anti-TROP-2 and anti-CEACAM6 antibodies in xenograftsexpressing their target antigens.

Milatuzumab-doxorubicin was most effective in the lymphoma model, whilein A-375 and Capan-1, only the milatuzumab-CL2A-SN-38 showed atherapeutic benefit. Despite much lower surface expression of CD74 thanTROP-2 or CEACAM6, milatuzumab-CL2A-SN-38 had similar efficacy inCapan-1 as anti-TROP-2-CL2A-SN-38, but in NCI-N87, the anti-CEACAM6 andanti-TROP-2 conjugates were superior. Studies in 2 hepatoma cell linesat a single dose level showed significant benefit over saline-treatedanimals, but not against an irrelevant IgG conjugate. CD74 is a suitabletarget for ADCs in some solid tumor xenografts, with efficacy largelyinfluenced by uniformity of CD74 expression, and with CL2A-linked SN-38conjugates providing the best therapeutic responses.

Introduction

CD74, referred to as invariant chain or Ii, is a type II transmembraneglycoprotein that associates with HLA-DR and inhibits the binding ofantigenic peptides to the class II antigen presentation structure. Itserves as a chaperone molecule, directing the invariant chain complexesto endosomes and lysosomes, an accessory molecule in the maturation of Bcells, using a pathway mediated by NF-kB, and in T-cell responses viainteractions with CD44 (Naujokas et al., 1993, Cell 74:257-68), and itis a receptor for the pro-inflammatory cytokine, macrophage migrationinhibitory factor (Leng et al., 2003, J Exp Med 197:1467-76), which isinvolved in activating cell proliferation and survival pathways.

In normal human tissues, CD74 is primarily expressed in B cells,monocytes, macrophages, dendritic cells, Langerhans cells, subsets ofactivated T cells, and thymic epithelium (not shown), and it isexpressed in over 90% of B-cell tumors (Burton et al., 2004, Clin CancerRes 10:6606-11; Stein et al., 2004, Blood 104:3705-11). Early studieshad conflicting data on whether CD74 is present on the membrane, in partbecause the antibodies to the invariant chain were specific for thecytoplasmic portion of the molecule, but also because there arerelatively few copies on the surface, and its half-life on the cellsurface is very short. Approximately 80% of the CD74 on the cell surfaceis associated with the MHC II antigen HLA-DR (Roche et al., 1993, PNASUSA 90:8581-85). Using the murine anti-CD74 antibody, LL1, the RajiBurkitt lymphoma cell line was estimated to have 4.8×10⁴ copies/cell,but because of rapid intracellular transit, ˜8×10⁶ antibody moleculeswere internalized and catabolized per day (Hansen et al., 1996, BiochemJ 320:293-300). Thus, CD74 internalization is highly dynamic, with theantibody being moved quickly from the surface and unloaded inside thecell, followed by CD74 re-expression on the surface. Fab′internalization occurs just as rapidly as IgG binding, indicating thatbivalent binding is not required. Later studies with a CDR-graftedversion of murine LL1, milatuzumab (hLL1), found that the antibody couldalter B-cell proliferation, migration, and adhesion molecule expression(Stein et al., 2004, Blood 104:3705-11; Qu et al., 2002, Proc Am AssocCancer Res 43:255; Frolich et al., 2012, Arthritis Res Ther 14:R54), butthe exceptional internalization properties of the anti-CD74 antibodymade it an efficient carrier for the intracellular delivery of cancertherapeutics (e.g., Griffiths et al., 2003, Clin Cancer Res 9:6567-71).Based on preclinical efficacy and toxicology results, Phase I clinicaltrials with milatuzumab-doxorubicin in multiple myeloma (Kaufman et al.,2008, ASH Annual Meeting Abstracts, 112:3697), as well as non-Hodgkinlymphoma and chronic lymphocytic leukemia, have been initiated.

Interestingly, CD74 also is expressed in non-hematopoietic cancers, suchas gastric, renal, urinary bladder, non-small cell lung cancers, certainsarcomas, and glioblastoma (e.g., Gold et al., 2010, Int J Clin ExpPathol 4:1-12), and therefore it may be a therapeutic target for solidtumors expressing this antigen. Since a milatuzumab-doxorubicinconjugate was highly active in models of hematological cancers, it was alogical choice for this assessment. However, we recently developedprocedures for coupling the highly potent topoisomerase I inhibitor,SN-38, to antibodies. SN-38 is the active form of irinotecan, whosepharmacology and metabolism are well known. These conjugates havenanomolar potency in solid tumor cell lines, and were found to be activewith antibodies that were not actively internalized. Prior studiesindicated a preference for a linker (CL2A) that allowed SN-38 todissociate from the conjugate in serum with a half-life of ˜1 day,rather than other linkers that were either more or less stable in serum.However, given milatuzumab's exceptional internalization capability, anew linker (CL2E) that is highly stable in serum, but can release SN-38when taken into the lysosome, was developed.

The current investigation examines the prospects for using these threemilatuzumab anti-CD74 conjugates, one with doxorubicin, and two SN-38conjugates, for effective therapy primarily against solid tumors.

Materials and Methods

Human Tumor Cell Lines. Raji Burkitt lymphoma, A-375 (melanoma), Capan-1(pancreatic adenocarcinoma), NCI-N87 (gastric carcinoma), Hep-G2hepatoma and MC/CAR myeloma cell lines were purchased from AmericanTissue Culture Collection (Manassas, Va.). HuH-7 hepatoma cell line waspurchased from Japan Health Science Research Resources Bank (Osaka,Japan). All cell lines were cultured in a humidified CO₂ incubator (5%)at 37° C. in recommended media containing 10% to 20% fetal-calf serumand supplements. Cells were passaged <50 times and checked regularly formycoplasma.

Antibodies and Conjugation Methods. Milatuzumab (anti-CD74 MAb),epratuzumab (anti-CD22), veltuzumab (anti-CD20), labetuzumab(anti-CEACAM5), hMN15 (anti-CEACAM6), and hRS7 (anti-TROP-2) arehumanized IgG₁ monoclonal antibodies. CL2A and CL2E linkers and theirSN-38 derivatives were prepared and conjugated to antibodies asdescribed in the Examples above. The milatuzumab-doxorubicin conjugateswere prepared as previously described (Griffiths et al., 2003, ClinCancer Res 9:6567-71). All conjugates were prepared by disulfidereduction of the IgG, followed by reaction with the correspondingmaleimide derivatives of these linkers. Spectrophotometric analysesestimated the drug:IgG molar substitution ratio was 5-7 (1.0 mg of theprotein contains ˜16 μg of SN-38 or 25 μg of doxorubicin equivalent).

In Vitro Cell Binding and Cytotoxicity. Assays to compare cell bindingof the unconjugated and conjugated milatuzumab to antigen-positive cellsand cytotoxicity testing used the MTS dye reduction method (Promega,Madison, Wis.).

Flow Cytometry and Immunohistology. Flow cytometry was performed in amanner that provided an assessment of only membrane-bound or membraneand cytoplasmic antigen. Immunohistology was performed onformalin-fixed, paraffin-embedded sections of subcutaneous tumorxenografts, staining without antigen retrieval methods, using antibodiesat 10 μg/mL that were revealed with an anti-human IgG conjugate.

In Vivo Studies. Female nude mice (4-8 weeks old) or female SCID mice (7weeks old) were purchased from Taconic (Germantown, N.Y.) and used aftera 1-week quarantine. All agents, including saline controls, wereadministered intraperitoneally twice-weekly for 4 weeks. Specific dosesare given in Results. Toxicity was assessed by weekly weightmeasurements. For the Raji Burkitt lymphoma model, SCID mice wereinjected intravenously with 2.5×10⁶Raji cells in 0.1 mL media. Five dayslater, animals received a single intravenous injection (0.1 mL) of theconjugate or saline (N=10/group). Mice were observed daily for signs ofdistress and paralysis, and were euthanized when either hind-limbparalysis developed, >15% loss of initial weight, or if otherwisemoribund (surrogate survival endpoints).

Subcutaneous tumors were measure by caliper in two dimensions, and thetumor volume (TV) calculated as L×w²/2, where L is the longest diameterand w is the shortest. Measurements were made at least once weekly, withanimals terminated when tumors grew to 1.0 cm³ (i.e., surrogate survivalend-point). The A-375 melanoma cell line (6×10⁶ cells in 0.2 mL) wasimplanted in nude mice and therapy was initiated when tumors averaged0.23±0.06 cm³ (N=8/group). Capan-1 was implanted subcutaneously in nudemice using a combination of tumor suspension from serially-passagedtumors (0.3 mL of a 15% w/v tumor suspension) combined with 8×10⁶ cellsfrom tissue culture. Treatments were initiated when TV averaged0.27±0.05 cm³ (N=10/group). NCI-N87 gastric tumor xenografts wereinitiated by injecting 0.2 mL of a 1:1 (v/v) mixture of matrigel and1×10⁷ cells from terminal culture subcutaneously. Therapy was startedwhen the TV averaged 0.249±0.045 cm³ (N=7/group). The same procedure wasfollowed for developing the Hep-G2 and HuH-7 hepatoma xenografts in nudemice. Therapy was started when Hep-G2 averaged 0.364±0.062 cm³(N=5/group) and HuH-7 averaged 0.298±0.055 cm³ (N=5/group).

Efficacy is expressed in Kaplan-Meier survival curves, using thesurrogate end-points mentioned above for determining the median survivaltimes. Analysis was performed by a log-rank (Mantel-Cox) test usingPrism GraphPad software (LaJolla, Calif.), with significance at P<0.05.

Results

CD74 Expression in Human Tumor Cell Lines and Xenografts. Six cell linesderived from 4 different solid tumor types were identified asCD74-positive based primarily on the analysis of permeabilized cells(Table 11), since the MFI of membrane-only CD74 in the solid tumor celllines very often was <2-fold higher than the background MFI (exceptA-375 melanoma cell line). Surface CD74 expression in Raji was >5-foldhigher than the solid tumor cell lines, but total CD74 in permeabilizedRaji cells was similar to most of the solid tumor cell lines.

TABLE 11 CD74 expression by flow cytometry expressed as mean fluorescentintensity (MFI) of milatuzumab-positive gated cells. Surface Surface andcytoplasmic hLL1 MFI Ratio hLL1 MFI Ratio Cell line (bkgd)^(a) hLL1:bkgd(bkgd)^(b) hLL1:bkgd Panc CA^(c) Capan-1 22 (12) 1.8 248 (5) 49.6Gastric Hs746T 17 (8)  2.1 144 (5) 28.8 NCI-N87 5 (4) 1.3 220 (6) 36.7Melanoma A-375 16 (3)  5.3 185 (6) 30.8 Hepatoma Hep-G2 9 (6) 1.5 156(5) 31.2 HuH-7 8 (5) 1.6 114 (4) 28.5 Lymphoma Raji 59 (3)  19.6 143 (5)28.6 ND, not done ^(a)Background MFI of cells incubated with GAH-FITConly.

Immunohistology showed Raji subcutaneous xenografts had a largelyuniform and intense staining, with prominent cell surface labeling (notshown). The Hep-G2 hepatoma cell line had the most uniform uptake of thesolid tumors, with moderately strong, but predominantly cytoplasmic,staining (not shown), followed by the A-375 melanoma cell line that hadsomewhat less uniform staining with more intense, yet mostlycytoplasmic, expression (not shown). The Capan-1 pancreatic (not shown)and NCI-N87 (not shown) gastric carcinoma cell lines had moderate(Capan-1) to intense (NCI-N87) CD74 staining, but it was not uniformlydistributed. The HuH-7 hepatoma cell line (not shown) had the leastuniform and the weakest staining.

Immunoreactivity of the Conjugates. K_(d) values for unconjugatedmilatuzumab, milatuzumab-CL2A-SN-38 and milatuzumab-CL2E-SN-38conjugates were not significantly different, averaging 0.77 nM, 0.59 nM,and 0.80 nM, respectively. K_(d) values for the unconjugated anddoxorubicin-conjugated milatuzumab measured in the MC/CAR multiplemyeloma cell line were 0.5±0.02 nM and 0.8±0.2 nM, respectively (Sapraet al., 2008, Clin Cancer Res 14:1888-96).

In Vitro Drug Release and Serum Stabilities of Conjugates. The releasemechanisms of SN-38 from the mercaptoethanol-capped CL2A and CL2Elinkers were determined in an environment partially simulating lysosomalconditions, namely, low pH (pH 5.0), and in the presence or absence ofcathepsin B. The CL2E-SN-38 substrate was inert at pH 5 in the absenceof the enzyme (not shown), but in the presence of cathepsin B, cleavageat the Phe-Lys site proceeded quickly, with a half-life of 34 min (notshown). The formation of active SN-38 requires intramolecularcyclization of the carbamate bond at the 10^(th) position of SN-38,which occurred more slowly, with a half-life of 10.7 h (not shown).

As expected, cathepsin B had no effect on the release of active SN-38 inthe CL2A linker. However, CL2A has a cleavable benzyl carbonate bond,releasing active SN-38 at a rate similar to the CL2E linker at pH 5.0,with a half-life of ˜10.2 h (not shown). The milatuzumab-doxorubicinconjugate, which has a pH-sensitive acylhydrazone bond, had a half-lifeof 7 to 8 h at pH 5.0 (not shown).

While all of these linkers release the drug at relatively similar ratesunder lysosomally-relevant conditions, they have very differentstabilities in serum. Milatuzumab-CL2A-SN-38 released 50% of free SN-38in 21.55±0.17 h (not shown), consistent with other CL2A-SN-38conjugates. The CL2E-SN-38 conjugate, however, was highly inert, with ahalf-life extrapolated to 2100 h. The milatuzumab-doxorubicin conjugatereleased 50% of the doxorubicin in 98 h, which was similar to 2 otherantibody-doxorubicin conjugates (not shown).

Cytotoxicity. A significant issue related to the evaluation of theseconjugates was the relative potency of free doxorubicin and SN-38 inhematopoietic and solid tumor cell lines. Our group previously reportedthat SN-38 was active in several B-cell lymphoma and acute leukemia celllines, with potencies ranging from 0.13 to 2.28 nM (Sharkey et al.,2011, Mol Cancer Ther 11:224-34). SN-38 potency in 4 of the solid tumorcell lines that were later used for in vivo therapy studies ranged from2.0 to 6 nM (not shown). Doxorubicin had a mixed response, with 3-4 nMpotency in the Raji lymphoma and the A-375 melanoma cell lines, but itwas nearly 10 times less potent against Capan-1, NCI-N87, and Hep G2cell lines. Other studies comparing the potency of SN-38 to doxorubicinfound: LS174T colon cancer, 18 vs. 18 (nM potency of SN-38 vs.doxorubicin, respectively); MDA-MB-231 breast cancer, 2 vs. 2 nM;SK-OV-4 ovarian cancer, 18 vs. 90 nM; Calu-3 lung adenocarcinoma, 32 vs.582 nM; Capan-2 pancreatic cancer, 37 vs. 221 nM; and NCI-H466 smallcell lung cancer, 0.1 vs. 2 nM. Thus, SN-38 was 5- to 20-fold morepotent than doxorubicin in 4 of these 6 cell lines, with similar potencyin LS174T and MDA-MB-231. Collectively, these data indicate thatdoxorubicin is less effective against solid tumors than SN-38, whileSN-38 appears to be equally effective in solid and hematopoietic tumors.

As expected, the 3 conjugate forms were often some order of magnitudeless potent than the free drug in vitro, since both drugs are expectedto be transported readily into the cells, while drug conjugates requireantibody binding to transport drug inside the cell (not shown). TheCL2A-linked SN-38 conjugate is an exception, since more than 90% of theSN-38 is released from the conjugate into the media over the 4-day assayperiod (Cardillo et al., 2011, Clin Cancer Res 17:3157-69; Sharkey etal., 2011, Mol Cancer Ther 11:224-34). Thus, even if the conjugate wasinternalized rapidly, it would be difficult to discern differencesbetween the free drug and the CL2A-linked drug.

The stable CL2E-linked SN-38 performed comparatively well in the Rajicell line, compared to free SN-38, but it had substantially (7- to16-fold) lower potency in the 4 solid tumor cell lines, suggesting therelatively low surface expression of CD74 may be playing a role inminimizing drug transport in these solid tumors. Themilatuzumab-doxorubicin conjugate had substantial differences in itspotency when compared to the free doxorubicin in all cell lines, whichwas of similar magnitude as the CL2E-SN-38 conjugates to free SN-38 inthe solid tumor cell lines.

In the 6 additional cell lines mentioned above, themilatuzumab-CL2A-SN-38 conjugate was 9- to 60-times more potent than themilatuzumab-doxorubicin conjugate (not shown), but again, this resultwas influenced largely by the fact that the CL2A-linked conjugatereleases most of its SN-38 into the media over the 4-day incubationperiod, whereas the doxorubicin conjugate would at most release 50% ofits drug over this same time. The CL2E-linked milatuzumab was notexamined in these other cell lines.

In Vivo Therapy of Human Tumor Xenografts. Previous in vivo studies withthe milatuzumab-doxorubicin or SN-38 conjugates prepared with variousantibodies had indicated they were efficacious at doses far lower thantheir maximum tolerated dose (Griffiths et al., 2003, Clin Cancer Res9:6567-71; Sapra et al., 2005, Clin Cancer Res 11:5257-64; Govindan etal., 2009, Clin Cancer Res 15:6052-61; Cardillo et al., 2011, ClinCancer Res 17:3157-69; Sharkey et al., 2011, Mol Cancer Ther 11:224-34),and thus in vivo testing focused on comparing similar, but fixed,amounts of each conjugate at levels that were well-tolerated.

Initial studies first examined the doxorubicin and SN-38 conjugates in adisseminated Raji model of lymphoma in order to gauge how themilatuzumab-doxorubicin conjugate compared to the 2 SN-38 conjugates(not shown). All specific conjugates were significantly better thannon-targeting labetuzumab-SN-38 conjugate or saline-treated animals,which had a median survival of only 20 days (P<0.0001). Despite in vitrostudies indicating as much as an 8-fold advantage for the SN-38conjugates in Raji, the best survival was seen with themilatuzumab-doxorubicin conjugates, where all animals given a single17.5 mg/kg (350 μg) dose and 7/10 animals given 2.0 mg/kg (40 μg) werealive at the conclusion of the study (day 112) (e.g., 17.5 mg/kg dosemilatuzumab-doxorubicin vs. milatuzumab-CL2A-SN-38, P=0.0012). Survivalwas significantly lower for the more stable CL2E-SN-38 conjugates(P<0.0001 and P=0.0197, 17.5 and 2.0 mg/kg doses for the CL2A vs. CL2E,respectively), even though in vitro studies suggested that bothconjugates would release active SN-38 at similar rates wheninternalized.

Five solid tumor cell lines were examined, starting with the A-375melanoma cell line, since it had the best in vitro response to bothdoxorubicin and SN-38. A-375 xenografts grew rapidly, withsaline-treated control animals having a median survival of only 10.5days (not shown). A 12.5 mg/kg (0.25 mg per animal) twice-weekly dose ofthe milatuzumab-CL2A-SN-38 conjugate extended survival to 28 days(P=0.0006), which was significantly better than the controlepratuzumab-CL2A-SN-38 conjugate having a median survival of 17.5 days(P=0.0089), with the latter not being significantly different from thesaline-treated animals (P=0.1967). The milatuzumab-CL2A conjugateprovided significantly longer survival than the milatuzumab-CL2E-SN-38conjugate (P=0.0014), which had the same median survival of 14 days asits control epratuzumab-CL2E-SN-38 conjugate. Despite giving a 2-foldhigher dose of the milatuzumab-doxorubicin than the SN-38 conjugates,the median survival was no better than the saline-treated animals (10.5days).

As with the A-375 melanoma model, in Capan-1, only the CL2A-linked SN-38conjugate was effective, with a median survival of 35 days,significantly different from untreated animals (P<0.036) (not shown),even at a lower dose (5 mg/kg; 100 μg per animal) (P<0.02). Neither themilatuzumab-CL2E nor the non-targeting epratuzumab-CL2A-SN-38conjugates, or a 2-fold higher dose of the milatuzumab-doxorubicinconjugate, provided any survival advantage (P=0.44 vs. saline). It isnoteworthy that in the same study with animals given the same dose ofthe internalizing anti-TROP-2 CL2A-SN-38 conjugate (hRS7-SN-38;IMMU-132), the median survival was equal to milatuzumab-CL2A-SN-38 (notshown). The hRS7-CL2A-SN-38 conjugate had been identified previously asan ADC of interest for treating a variety of solid tumors (Cardillo etal., 2011, Clin Cancer Res 17:3157-69). The MFI for surface-binding hRS7on Capan-1 was 237 (not shown), compared to 22 for milatuzumab (seeTable 11). Thus, despite having a substantially lower surface antigenexpression, the milatuzumab-CL2A-SN-38 conjugate performed as well asthe hRS7-CL2A-SN-38 conjugate in this model.

With the milatuzumab-doxorubicin conjugate having inferior therapeuticresults in 2 of the solid tumor xenografts, the focus shifted to comparethe milatuzumab-SN-38 conjugates to SN-38 conjugates prepared with otherhumanized antibodies against TROP-2 (hRS7) or CEACAM6 (hMN-15), whichare more highly expressed on the surface of many solid tumors(Blumenthal et al., 2007, BMC Cancer 7:2; Stein et al., 1993, Int JCancer 55:938-46). Three additional xenograft models were examined.

In the gastric tumor model, NCI-N87, animals given 17.5 mg/kg/dose (350μg) of milatuzumab-CL2A-SN-38 provided some improvement in survival, butit failed to meet statistical significance compared to thesaline-treated animals (31 vs. 14 days; P=0.0760) or to the non-bindingveltuzumab anti-CD20-CL2A-SN39 conjugate (21 days; P=0.3128) (notshown). However, the hRS7- and hMN-15-CL2A conjugates significantlyimproved the median survival to 66 and 63 days, respectively (P=0.0001).The MFI for surface-expressed TROP-2 and CEACAM6 were 795 and 1123,respectively, much higher than CD74 that was just 5 (see Table 11).Immunohistology showed a relatively intense cytoplasmic expression ofCD74 in the xenograft of this cell line, but importantly it wasscattered, appearing only in defined pockets within the tumor (notshown). CEACAM6 and TROP-2 were more uniformly expressed than CD74 (notshown), with CEACAM6 being more intensely present both cytoplasmicallyand on the membrane, and TROP-2 primarily found on the membrane. Thus,the improved survival with the anti-CEACAM6 and anti-TROP-2 conjugatesmost likely reflects both higher antigen density and more uniformexpression in NCI-N87.

In the Hep-G2 hepatoma cell line (not shown), immunohistology showed avery uniform expression with moderate cytoplasmic staining of CD74, andflow cytometry indicated a relatively low surface expression (MFI=9).The MFI with hMN-15 was 175 and immunohistology showed a fairly uniformmembrane and cytoplasmic expression of CEACAM6, with isolated pockets ofvery intense membrane staining (not shown). A study in animals bearingHep-G2 xenografts found the milatuzumab-CL2A-SN-38 extended survival to45 days compared to 21 days in the saline-treated group (P=0.0048),while the hMN-15-CL2A-SN-38 conjugate improved survival to 35 days.There was a trend favoring the milatuzumab conjugate overhMN-15-CL2A-SN-38, but it did not achieve statistical significance (46vs. 35 days; P=0.0802). However, the non-binding veltuzumab-CL2A-SN-38conjugate provided a similar survival advantage as the milatuzumabconjugate. We previously observed therapeutic results with non-bindingconjugates could be similar to the specific CL2A-linked conjugate,particularly at higher protein doses, but titration of the specific andcontrol conjugates usually revealed selectively. Thus, neither of thespecific conjugates provided a selective therapeutic advantage at thesedoses in this cell line.

Another study using the HuH-7 hepatoma cell line (not shown), which hadsimilar surface expression, but slightly lower cytoplasmic levels asHep-G2 (see Table 11), found the hMN-15-SN-38 conjugate providing alonger (35 vs. 18 days), albeit not significantly different, survivaladvantage than the milatuzumab-CL2A conjugate (P=0.2944). While both thehMN-15 and milatuzumab conjugates were significantly better than thesaline-treated animals (P=0.008 and 0.009, respectively), again, neitherconjugate was significantly different from the non-targetedveltuzumab-SN-38 conjugate at this dose level (P=0.4602 and 0.9033,respectively). CEACAM6 surface expression was relatively low in thiscell line (MFI=81), and immunohistology showed that both CD74 (notshown) and CEACAM6 (not shown) were very faint and highly scattered.

Discussion

The antibody-drug conjugate (ADC) approach for tumor-selectivechemotherapy is an area of considerable current interest (e.g., Govindanet al., 2012, Expert Opin Biol Ther 12:873-90; Sapra et al., 2011,Expert Opin Biol Ther 20:1131-49. The recent clinical successes (Pro etal., 2012, Expert Opin Biol Ther 12:1415-21; LoRusso et al., 2011, ClinCancer Res 17:437-47) have occurred in a large part with the adoption ofsupertoxic drugs in place of the conventional chemotherapeutic agentsthat had been used previously. However, target selection, the antibody,and the drug linker are all factors that influence optimal performanceof an ADC. For example, in the case of trastuzumab-DM1, HER2 is abundanton tumors expressing this antigen, the antibody is internalized, and theantibody itself has anti-tumor activity, all of which could combine toenhance therapeutic outcome. In stark contrast, CD74 is expressed at amuch lower level on the surface of cells, but its unique internalizationand surface re-expression properties has allowed a milatuzumab anti-CD74ADC to be effective in hematopoietic cancer xenograft models even with amoderately toxic drug, such as doxorubicin (Griffiths et al., 2003, ClinCancer Res 9:6567-71; Sapra et al., 2005, Clin Cancer Res 11:5257-64).Although doxorubicin is used more frequently in hematopoietic cancers,while SN-38 and other camptothecins are administered to patients withsolid tumors, we decided to assess the utility of doxorubicin and SN-38conjugates of milatuzumab in solid tumors. The milatuzumab-doxorubicinconjugate was effective in xenograft models of various hematologicalcancers, leading to its clinical testing (NCT01101594 and NCT01585688),while several SN-38 conjugates were effective in solid and hematologicaltumor models, leading to 2 new SN-38 conjugates being pursued in Phase Iclinical trials of colorectal and diverse epithelial cancers(NCT01270698 and NCT01631552).

In vitro, unconjugated doxorubicin and SN-38 had similar potency asdoxorubicin against the Raji lymphoma cell line, but SN-38 was morepotent in a number of different solid tumor cell lines. Interestingly,in vivo, the milatuzumab-doxorubicin conjugate provided the bestresponse in Raji as compared to the milatuzumab-SN-38 conjugates.However, in Capan-1 and A-375, milatuzumab-doxorubicin was lesseffective than the CL2A-linked SN-38 milatuzumab conjugate, even thoughin vitro testing had indicated that A-375 was equally sensitive to freedoxorubicin as to free SN-38. Two other cell lines, MDA-MB-231 breastcancer and LS174T colon cancer, also had similar potency with freedoxorubicin as SN-38 in vitro, but since in vitro testing indicatedSN-38 was equally effective in solid and hematological cancers, and withSN-38 having a 5- to 20-fold higher potency than doxorubicin in mostsolid tumor cell lines evaluated, we decided to focus on the 2milatuzumab-SN-38 conjugates for solid tumor therapy. However, to bettergauge the utility of the milatuzumab-SN-38 conjugates, we included acomparative assessment to SN-38 ADCs prepared with antibodies againstother antigens that are present in a variety of solid tumors.

We previously had investigated therapeutic responses with theinternalizing hRS7 anti-TROP-2 CL2A-linked SN-38 conjugate in theCapan-1 cell line (Cardillo et al., 2011, Clin Cancer Res 17:3157-69),and therefore the efficacy of milatuzumab and hRS7 SN-38 conjugates werecompared. In this study, both conjugates significantly improved survivalcompared to control antibodies, with the CL2A-linked SN-38 conjugates ofeach being superior to the CL2E-linked conjugates. Since flow cytometryhad indicated TROP-2 expression was higher than CD74 in Capan-1, thisresult suggested that the transport capabilities of CD74, which wereknown to be exceptional, were more efficient than TROP-2. However, it iswell known that antigen accessibility (i.e., membrane vs. cytoplasm,physiological and “binding-site” barriers) and distribution among cellswithin a tumor are critical factors influencing every form of targetedtherapy, particularly those that depend on adequate intracellulardelivery of a product to individual cells (Thurber et al., 2008, AdvDrug Del Rev 60:1421-34). In situations where the antigen is notuniformly expressed in all cells within the tumor, having a targetedagent that slowly releases its payload after localizing in the tumor,such as the CL2A-linked conjugates, would allow the drug to diffuse tonon-targeted bystander cells, thereby enhancing its efficacy range.Indeed, high antigen expression could potentially impede tumorpenetration as per the binding-site barrier effect, but theextracellular release mechanism could provide a mechanism for the drugto diffuse within the tumor. This mechanism also is thought to aid theefficacy of other conjugates that we have examined using poorlyinternalizing antibodies, such as anti-CEACAM5 and the anti-CEACAM6 usedherein. Conjugates based on milatuzumab rely more heavily on theantibody's direct interaction with the tumor cell, taking advantage ofCD74's rapid internalization and re-expression that can compensate forits lower abundance on the surface of cells. However, this advantagewould be mitigated when CD74 is highly scattered within the tumor, andwithout a mechanism to retain the conjugate within the tumor, thebenefit of the drug's slow release from the conjugate would be lost. Aprevious review of human gastrointestinal tumors by our group suggeststhat they often have a high level of expression with good uniformity(Gold et al., 2010, Int J Clin Exp Pathol 4:1-12).

During our initial assessment of suitable linkers for SN-38, a number ofdifferent derivatives were examined, including a ‘CL2E’-like linker thatwas designed to be coupled at the 20-hydroxyl position of SN-38, similarto the CL2A linker. However, that antibody conjugate lacked sufficientantitumor activity and was not pursued. Given the exceptionalinternalization properties of milatuzumab, we decided to revisit theSN-38-linker chemistry, with the hypothesis that the rapidinternalization of a CD74 conjugate would enhance drug loading of a morestable conjugate. We surmised that if the leaving group was phenolic,this could promote cyclization, and therefore, the CL2E-linker wasdesigned to join at the phenolic 10-position of SN-38.

At the onset, the CL2E-linked SN-38 conjugate had a promisingly similarIC₅₀ as the CL2A conjugate in the Raji cell line, which was consistentwith the view that if rapidly internalized, both conjugates wouldrelease the active form of SN-38 at approximately the same rate.However, as already mentioned, the in vitro activity of the CL2Aconjugate is influenced largely by the release of SN-38 into the media,and does not necessarily reflect uptake by the intact conjugate. Whenthe CL2E-linked conjugate was found to be much less potent in the solidtumor cell lines than the CL2A conjugate, this suggested that the lowersurface expression of CD74 affected the internalization of SN-38 viamilatuzumab binding. However, when in vivo studies in Raji showed themilatuzumab-CL2A-SN-38 was superior to the CL2E conjugate, some otherfactor had to be considered that would affect CL2E's efficacy. Onepossible explanation is that the linker design in CL2E-SN-38 leaves the20-position of the drug underivatized, rendering the lactone groupsusceptible to ring-opening. Indeed, studies with irinotecan have shownSN-38's potency is diminished by a number of factors, with the lactonering opening to the carboxylate form possessing only 10% of the potencyof the intact lactone form. In contrast, the CL2A-linked SN-38 isderivatized at the 20-hydroxyl position, a process that stabilizes thelactone group in camptothecins under physiological conditions.Therefore, SN-38's lactone ring is likely protected from cleavage in theCL2A, but not the CL2E conjugate. Thus, the destabilization of thelactone ring could have contributed to CL2E's diminished efficacy invivo. Since the in vitro stability studies and the analysis of serumstability were performed under acidic conditions, we do not have adirect measure of the carboxylate form of SN-38 in either of theseconjugates.

In conclusion, in vitro and in vivo results indicate that themilatuzumab-doxorubicin conjugate is superior to the CL2A-SN-38conjugate in the Raji lymphoma cell line, which may reflect the improvedstability of the doxorubicin conjugate compared to the CL2A one.However, the finding that the CL2A-SN-38 conjugate was more effectivethan the highly stable CL2E-SN-38 conjugate suggests that other issues,potentially related to activation of the drug or cell linesensitivities, may be at play.

CD74 has multiple roles in cell biology; in antigen-presenting cells, itmay have a more dominant role in processing antigenic peptides, where issolid tumors, its role might be related more to survival. Thesedifferent roles could affect intracellular trafficking and processing.Alternatively, the lower efficacy of the CL2E-linked SN-38 could reflectdrug inactivation by lactone ring-opening in SN-38, implicating theimportance of the specific linker. Finally, in the solid tumor models,antigen accessibility appears to have a dominant role in definingmilatuzumab-CL2A-SN-38's potency when measured against conjugatesprepared with other internalizing (hRS7) or poorly internalizingantibodies (hMN15) that were more accessible (surface expressed) andabundant. We suspect this finding is universal for targeted therapies,but these studies have at least shown that the unique internalizationproperties of a CD74-targeted agent can provide significant efficacyeven when surface expression of the target antigen is minimal.

Example 15 Use of hRS7-SN-38 (IMMU-132) to Treat Therapy-refractiveMetastatic Colonic Cancer (mCRC)

The patient was a 62-year-old woman with mCRC who originally presentedwith metastatic disease in January 2012. She had laparoscopic ilealtransverse colectomy as the first therapy a couple of weeks afterdiagnosis, and then received 4 cycles of FOLFOX (leucovorin,5-fluorouracil, oxaliplatin) chemotherapy in a neoadjuvant setting priorto right hepatectomy in March 2012 for removal of metastatic lesions inthe right lobe of the liver. This was followed by an adjuvant FOLFOXregimen that resumed in June, 2012, for a total of 12 cycles of FOLFOX.In August, oxaliplatin was dropped from the regimen due to worseningneurotoxicity. Her last cycle of 5-FU was on 09/25/12.

CT done in January 2013 showed metastases to liver. She was thenassessed as a good candidate for enrollment to IMMU-132(hRS7-CL2A-SN-38) investigational study. Comorbidities in her medicalhistory include asthma, diabetes mellitus, hypertension,hypercholesteremia, heart murmur, hiatal hernia, hypothyroidism, carpeltunnel syndrome, glaucoma, depression, restless leg syndrome, andneuropathy. Her surgical history includes tubo-ligation (1975),thyroidectomy (1983), cholescystectomy (2001), carpel tunnel release(2008), and glaucoma surgery.

At the time of entry into this trial, her target lesion was a 3.1-cmtumor in the left lobe of the liver. Non-target lesions included severalhypo-attenuated masses in the liver. Her baseline CEA was 781 ng/m.

After the patient signed the informed consent, IMMU-132 was given on aonce-weekly schedule by infusion for 2 consecutive weeks, then a rest ofone week, this constituting a treatment cycle. These cycles wererepeated as tolerated. The first infusion of IMMU-132 (8 mg/kg) wasstarted on Feb. 15, 2013, and completed without notable events. Sheexperienced nausea (Grade 2) and fatigue (Grade 2) during the course ofthe first cycle and has been continuing the treatment since then withoutmajor adverse events. She reported alopecia and constipation in March2013. The first response assessment done (after 6 doses) on Apr. 8, 2013showed a shrinkage of target lesion by 29% by computed tomography (CT).Her CEA level decreased to 230 ng/ml on Mar. 25, 2013. In the secondresponse assessment (after 10 doses) on May 23, 2013, the target lesionshrank by 39%, thus constituting a partial response by RECIST criteria.She has been continuing treatment as of Jun. 14, 2013, receiving 6cycles constituting 12 doses of hRS7-CL2A-SN-38 (IMMU-132) at 8 mg/kg.Her overall health and clinical symptoms improved considerably sincestarting this investigational treatment.

Example 16 Use of hRS7-SN-38 (IMMU-132) to Treat Therapy-refractiveMetastatic Breast Cancer

The patient was a 57-year-old woman with stage IV, triple-negative,breast cancer (ER/PR negative, HER-neu negative), originally diagnosedin 2005. She underwent a lumpectomy of her left breast in 2005, followedby Dose-Dense ACT in adjuvant setting in September 2005. She thenreceived radiation therapy, which was completed in November. Localrecurrence of the disease was identified when the patient palpated alump in the contralateral (right) breast in early 2012, and was thentreated with CMF (cyclophosphamide, methotrexate, 5-fluorouracil)chemotherapy. Her disease recurred in the same year, with metastaticlesions in the skin of the chest wall. She then received acarboplatin+TAXOL® chemotherapy regimen, during which thrombocytopeniaresulted. Her disease progressed and she was started on weeklydoxorubicin, which was continued for 6 doses. The skin disease also wasprogressing. An FDG-PET scan on 09/26/12 showed progression of diseaseon the chest wall and enlarged, solid, axillary nodes. The patient wasgiven oxycodone for pain control.

She was given IXEMPRA® from October 2012 until February 2013 (every 2weeks for 4 months), when the chest wall lesion opened up and bled. Shewas then put on XELODA®, which was not tolerated well due to neuropathyin her hands and feet, as well as constipation. The skin lesions wereprogressive and then she was enrolled in the IMMU-132 trial after givinginformed consent. The patient also had a medical history ofhyperthyroidism and visual disturbances, with high risk of CNS disease(however, brain MM was negative for CNS disease). At the time ofenrollment to this trial, her cutaneous lesions (target) in the rightbreast measured 4.4 cm and 2.0 cm in the largest diameter. She hadanother non-target lesion in the right breast and one enlarged lymphnode each in the right and left axilla.

The first IMMU-132 infusion (12 mg/kg) was started on Mar. 12, 2013,which was tolerated well. Her second infusion was delayed due to Grade 3absolute neutrophil count (ANC) reduction (0.9) on the scheduled day ofinfusion, one week later. After a week delay and after receivingNEULASTA®, her second IMMU-132 was administered, with a 25% dosereduction at 9 mg/kg. Thereafter she has been receiving IMMU-132 onschedule as per protocol, once weekly for 2 weeks, then one week off.Her first response assessment on May 17, 2013, after 3 therapy cycles,showed a 43% decrease in the sum of the long diameter of the targetlesions, constituting a partial response by RECIST criteria. She iscontinuing treatment at the 9 mg/kg dose level. Her overall health andclinical symptoms improved considerably since she started treatment withIMMU-132.

Example 17 Use of hRS7-SN-38 (IMMU-132) to Treat Refractory, Metastatic,Non-small Cell Lung Cancer

This is a 60-year-old man diagnosed with non-small cell lung cancer. Thepatient is given chemotherapy regimens of carboplatin, bevacizumab for 6months and shows a response, and then after progressing, receivesfurther courses of chemotherapy with carboplatin, etoposide, TAXOTERE®,gemcitabine over the next 2 years, with occasional responses lasting nomore than 2 months. The patient then presents with a left mediastinalmass measuring 6.5×4 cm and pleural effusion.

After signing informed consent, the patient is given IMMU-132 at a doseof 18 mg/kg every other week. During the first two injections, briefperiods of neutropenia and diarrhea are experienced, with 4 bowelmovements within 4 hours, but these resolve or respond to symptomaticmedications within 2 days. After a total of 6 infusions of IMMU-132, CTevaluation of the index lesion shows a 22% reduction, just below apartial response but definite tumor shrinkage. The patient continueswith this therapy for another two months, when a partial response of 45%tumor shrinkage of the sum of the diameters of the index lesion is notedby CT, thus constituting a partial response by RECIST criteria.

Example 18 Use of hRS7-CL2A-SN-38 (IMMU-132) to Treat Refractory,Metastatic, Small-cell Lung Cancer

This is a 65-year-old woman with a diagnosis of small-cell lung cancer,involving her left lung, mediastinal lymph nodes, and MM evidence of ametastasis to the left parietal brain lobe. Prior chemotherapy includescarboplatin, etoposide, and topotecan, but with no response noted.Radiation therapy also fails to control her disease. She is then givenIMMU-132 at a dose of 18 mg/kg once every three weeks for a total of 5infusions. After the second dose, she experiences hypotension and aGrade 2 neutropenia, which improve before the next infusion. After thefifth infusion, a CT study shows 13% shrinkage of her target left lungmass. Mill of the brain also shows a 10% reduction of this metastasis.She continues her IMMU-132 dosing every 3 weeks for another 3 months,and continues to show objective and subjective improvement of hercondition, with a 25% reduction of the left lung mass and a 21%reduction of the brain metastasis.

Example 19 Therapy of a Gastric Cancer Patient with Stage IV MetastaticDisease with hRS7-CL2A-SN-38 (IMMU-132)

This patient is a 60-year-old male with a history of smoking and periodsof excessive alcohol intake over a 40-year-period. He experiences weightloss, eating discomfort and pain not relieved by antacids, frequentabdominal pain, lower back pain, and most recently palpable nodes inboth axilla. He seeks medical advice, and after a workup is shown tohave an adenocarcinoma, including some squamous features, at thegastro-esophageal junction, based on biopsy via a gastroscope.Radiological studies (CT and FDG-PET) also reveal metastatic disease inthe right and left axilla, mediastinal region, lumbar spine, and liver(2 tumors in the right lobe and 1 in the left, all measuring between 2and 4 cm in diameter). His gastric tumor is resected and he is then puton a course of chemotherapy with epirubicin, cisplatin, and5-fluorouracil. After 4 months and a rest period of 6 weeks, he isswitched to docetaxel chemotherapy, which also fails to control hisdisease, based on progression confirmed by CT measurements of themetastatic tumors and some general deterioration.

The patient is then given therapy with IMMU-132 (hRS7-CL2A-SN-38) at adose of 10 mg/kg infused every-other-week for a total of 6 doses, afterwhich CT studies are done to assess status of his disease. Theseinfusions are tolerated well, with some mild nausea and diarrhea,controlled with symptomatic medications. The CT studies reveal that thesum of his index metastatic lesions has decreased by 28%, so hecontinues on this therapy for another 5 courses. Follow-up CT studiesshow that the disease remains about 35% reduced by RECIST criteria fromhis baseline measurements prior to IMMU-132 therapy, and his generalcondition also appears to have improved, with the patient regaining anoptimistic attitude toward his disease being under control.

Example 20 Therapy of Advanced Colon Cancer Patient Refractory to PriorChemo-immunotherapy, Using Only IMMU-130 (Labetuzumab-CL2A-SN-38)

The patient was a 50-year-old man with a history of stage-IV metastaticcolonic cancer, first diagnosed in 2008 and given a colectomy andpartial hepatectomy for the primary and metastatic colonic cancers,respectively. He then received chemotherapy, as indicated FIG. 8, whichincluded irinotecan, oxaliplatin, FOLFIRINOX (5-fluoruracil, leucovorin,irinotecan, oxaliplatin), and bevacizumab, as well as bevacizumabcombined with 5-fluorouracil/leucovorin, for almost 2 years. Thereafter,he was given courses of cetuximab, either alone or combined with FOLFIRI(leucovorin, 5-flurouracil, irinotecan) chemotherapy during the nextyear or more. In 2009, he received radiofrequency ablation therapy tohis liver metastasis while under chemo-immunotherapy, and in late 2010he underwent a wedge resection of his lung metastases, which wasrepeated a few months later, in early 2011. Despite havingchemo-immunotherapy in 2011, new lung metastases appeared at the end of2011, and in 2012, both lung and liver metastases were visualized. Hisbaseline plasma carcinoembryonic antigen (CEA) titer was 12.5 ng/mL justbefore undergoing the antibody-drug therapy with IMMU-130. The indexlesions chosen by the radiologist for measuring tumor size change bycomputed tomography were the mid-lobe of the right lung and the livermetastases, both totaling 91 mm as the sum of their longest diameters atthe baseline prior to IMMU-130 (anti-CEACAM5-CL2A-SN-38) therapy.

This patient received doses of 16 mg/kg of IMMU-130 by slow IV infusionevery other week for a total of 17 treatment doses. The patienttolerated the therapy well, having only a grade 1 nausea, diarrhea andfatigue after the first treatment, which occurred after treatments 4 and5, but not thereafter, because he received medication for theseside-effects. After treatment 3, he did show alopecia (grade 2), whichwas present during the subsequent therapy. The nausea, diarrhea, andoccasional vomiting lasted only 2-3 days, and his fatigue after thefirst infusion lasted 2 weeks. Otherwise, the patient tolerated thetherapy well. Because of the long duration of receiving this humanized(CDR-grafted) antibody conjugated with SN-38, his blood was measured foranti-labetuzumab antibody, and none was detected, even after 16 doses.

The first computed tomography (CT) measurements were made after 4treatments, and showed a 28.6% change from the sum of the measurementsmade at baseline, prior to this therapy, in the index lesions. After 8treatments, this reduction became 40.6%, thus constituting a partialremission according to RECIST criteria. This response was maintained foranother 2 months, when his CT measurements indicated that the indexlesions were 31.9% less than the baseline measurements, but somewhathigher than the previous decrease of 40.6% measured. Thus, based oncareful CT measurements of the index lesions in the lung and liver, thispatient, who had failed prior chemotherapy and immunotherapy, includingirinotecan (parent molecule of SN-38), showed an objective response tothe active metabolite of irintotecan (or camptotechin), SN-38, whentargeted via the anti-CEACAM5 humanized antibody, labetuzumab (hMN-14).It was surprising that although irinotecan (CPT-11) acts by releasingSN-38 in vivo, the SN-38 conjugated anti-CEACAM5 antibody provedeffective in a colorectal cancer patient by inducing a partial responseafter the patient earlier failed to respond to his lastirinotecan-containing therapy. The patient's plasma CEA titer reductionalso corroborated the CT findings: it fell from the baseline level of12.6 ng/mL to 2.1 ng/mL after the third therapy dose, and was between1.7 and 3.6 ng/mL between doses 8 and 12. The normal plasma titer of CEAis usually considered to be between 2.5 and 5.0 ng/mL, so this therapyeffected a normalization of his CEA titer in the blood.

Example 21 Therapy of a Patient with Advanced Colonic Cancer withIMMU-130

This patient is a 75-year-old woman initially diagnosed with metastaticcolonic cancer (Stage IV). She has a right partial hemicolectomy andresection of her small intestine and then receives FOLFOX, FOLFOX+bevacizumab, FOLFIRI+ramucirumab, and FOLFIRI+cetuximab therapies for ayear and a half, when she shows progression of disease, with spread ofdisease to the posterior cul-de-sac, omentum, with ascites in her pelvisand a pleural effusion on the right side of her chest cavity. Herbaseline CEA titer just before this therapy is 15 ng/mL. She is given 6mg/kg IMMU-130 (anti-CEACAM5-CL2A-SN-38) twice weekly for 2 consecutiveweeks, and then one week rest (3-week cycle), for more than 20 doses,which is tolerated very well, without any major hematological ornon-hematological toxicities. Within 2 months of therapy, her plasma CEAtiter shrinks modestly to 1.3 ng/mL, but at the 8-week evaluation sheshows a 21% shrinkage of the index tumor lesions, which increases to a27% shrinkage at 13 weeks. Surprisingly, the patient's ascites andpleural effusion both decrease (with the latter disappearing) at thistime, thus improving the patient's overall status remarkably. Thepatient continues her investigational therapy.

Example 22 Gastric Cancer Patient with Stage IV Metastatic DiseaseTreated with IMMU-130

The patient is a 52-year-old male who sought medical attention becauseof gastric discomfort and pain related to eating for about 6 years, andwith weight loss during the past 12 months. Palpation of the stomacharea reveals a firm lump which is then gastroscoped, revealing anulcerous mass at the lower part of his stomach. This is biopsied anddiagnosed as a gastric adenocarcinoma. Laboratory testing reveals nospecific abnormal changes, except that liver function tests, LDH, andCEA are elevated, the latter being 10.2 ng/mL. The patent then undergoesa total-body PET scan, which discloses, in addition to the gastrictumor, metastatic disease in the left axilla and in the right lobe ofthe liver (2 small metastases). The patient has his gastric tumorresected, and then has baseline CT measurements of his metastatictumors. Four weeks after surgery, he receives 3 courses of combinationchemotherapy consisting of a regimen of cisplatin and 5-fluorouracil(CF), but does not tolerate this well, so is switched to treatment withdocetaxel. It appears that the disease is stabilized for about 4 months,based on CT scans, but then the patient's complaints of further weightloss, abdominal pain, loss of appetite, and extreme fatigue causerepeated CT studies, which show increase in size of the metastases by asum of 20% and a suspicious lesion at the site of the original gastricresection.

The patient is then given experimental therapy with IMMU-130(anti-CEACAM5-CL2A-SN-38) on a weekly schedule of 8 mg/kg. He toleratesthis well, but after 3 weeks shows a grade 2 neutropenia and grade 1diarrhea. His fourth infusion is postponed by one week, and then theweekly infusions are reinstituted, with no evidence of diarrhea orneutropenia for the next 4 injection. The patient then undergoes a CTstudy to measure his metastatic tumor sizes and to view the originalarea of gastric resection. The radiologist measures, according to RECISTcriteria, a decrease of the sum of the metastatic lesions, compared tobaseline prior to IMMU-130 therapy, of 23%. There does not seem to beany clear lesion in the area of the original gastric resection. Thepatient's CEA titer at this time is 7.2 ng/mL, which is much reducedfrom the pre-IMMU-130 baseline value of 14.5 ng/mL. The patientcontinues on weekly IMMU-130 therapy at the same dose of 8.0 mg/kg, andafter a total of 13 infusions, his CT studies show that one livermetastasis has disappeared and the sum of all metastatic lesions isdecreased by 41%, constituting a partial response by RECIST. Thepatient's general condition improves and he resumes his usual activitieswhile continuing to receive a maintenance therapy of 8 mg/kg IMMU-130every third week for another 4 injections. At the last measurement ofblood CEA, the value is 4.8 ng/mL, which is within the normal range fora smoker, which is the case for this patient.

Example 23 Therapy of Relapsed Triple-negative Metastatic Breast Cancerwith hMN-15-CL2A-SN-38

A 58-year-old woman with triple-negative metastatic breast cancerformerly treated with bevacizumab plus paclitaxel, without response,presents with metastases to several ribs, lumbar vertebrae, a solitarylesion measuring 3 cm in diameter in her left lung, with considerablebone pain and fatigue. She is given an experimental therapy with theanti-CEACAM6 humanized monoclonal antibody, hMN-15 IgG, conjugated with6 molecules of SN-38 per IgG. She is given an infusion of 12 mg/kg everythird week, repeated for 4 doses, as a course of therapy. Except fortransient grade 2 neutropenia and some initial diarrhea, she toleratesthe therapy well, which is then repeated, after a rest of 2 months, foranother course. Radiological examination indicates that she has partialresponse by RECIST criteria, because the sum of the diameters of theindex lesions decrease by 39%. Her general condition, including bonepain, also improves, and she returns to almost the same level ofactivity as prior to her illness.

Example 24 Therapy of Relapsed, Generally Refractive, Metastatic ColonicCarcinoma with hMN-15-SN-38

A 46-year-old woman has Stage IV metastatic colonic cancer, with a priorhistory of resection of the primary lesion that also had synchronousliver metastases to both lobes of the liver, as well as a single focusof spread to the right lung; these metastases measured, by CT, between 2and 5 cm in diameter. She undergoes various courses of chemotherapy overa period of 3 years, including 5-fluorouracil, leucovorin, irinotecan,oxaliplatin, cetuximab, and bevacizumab. On two occasions, there isevidence of stabilization of disease or a short-term response, but noreduction of 30% or more of her measured lesions. Her plasma CEA titerat baseline prior to hMN-15-CL2A-SN-38 therapy is 46 ng/mL, and hertotal index lesions measure a sum of 92 mm.

Therapy with hMN-15-CL2A-SN-38 is instituted at 12 mg/kg weekly for 2weeks, with a rest period of one week thereafter, within a 21-day cycle.This cycle is repeated 3 times, with only transient neutropenia andgastrointestinal side effects (nausea, vomiting, diarrhea).Surprisingly, despite failing to respond to FOLFIRI therapy (whichincludes irinotecan, or CPT-11), the patient shows a partial response byRECIST criteria after completing her therapy. She is then placed on amaintenance schedule of this therapy at a dose of 16 mg/kg once everymonth for the next 6 months. Followup scans show that her diseaseremains under control as a partial response (PR), and the patient isgenerally in good condition with a 90% Kaaarnofsky performance status.

Example 25 Colonic Cancer Patient with Stage IV Metastatic DiseaseTreated with Anti-CSAp-SN-38 Conjugate

This patient presents with colonic cancer metastases to the left lobe ofthe liver and to both lungs, after having a resection of a 9-cm sigmoidcolon adenocarinoma, followed by chemo-immunotherapy with FOLIFIRI andcetuximab for 6 months, and then FOLFOX followed by bevacizumab for anadditional period of about 9 months. Ten months after the initialresection and then commencement of therapy, the stable disease thoughtto be present shows progression by the lesions growing and a newmetastasis appearing in the left adrenal gland. Her plasma CEA at thistime is 52 ng/mL, and her general condition appears to havedeteriorated, with abdominal pains, fatigue, and borderline anemia,suggesting possibly internal bleeding.

She is now given a CL2A-SN-38 conjugate of hMu-9 (anti-CSAp) antibody ata dose of 12 mg/kg weekly for two weeks, with one week rest, as atreatment cycle, and then repeated for additional treatment cycles,measuring her blood counts every week and receiving atropine medicationto gastrointestinal reactions. Grade 2 alopecia is noted after the firsttreatment cycle, but only a Grade 1 neutropenia. After 3 treatmentcycles, her plasma CEA titer is reduced to 19 ng/ml, and at this timeher CT measurements show a decrease of the index lesions in the liverand lungs by 24.1%. After an additional 3 courses of therapy, she showsa CT reduction of the index lesions of 31.4%, and a decrease in the sizeof the adrenal mass by about 40%. This patient is considered to beresponding to anti-CSAp-CL2A-SN-38 antibody-drug therapy, and continueson this therapy. Her general condition appears to be improved, with lessfatigue, no abdominal pain or discomfort, and generally more energy.

Example 26 Treatment of Breast Cancer with Anti-CEACAM6-CL2A-SN-38Immunoconjugate

This patient has triple-negative (does not express estrogen receptor,progesterone receptor or Her2/neu) metastatic breast cancer that isrelapsed after several different therapies over the past 3 years. Shepresents with several small tumors in both lungs, as well as metastasesto her C4, C5, T2, and T3 vertebrae, as well as the several ribsbilaterally. She is under standard therapy for her osteolytic lesions,and now begins treatment with hMN-15-CL2A-SN-38 at a dose of 16 mg/kgonce-weekly for 3 weeks, with a pause of one week, and then resumes this3-weekly-cycle therapy two more times. At 2 weeks post therapy, CT scansare performed to evaluate response, and it is noted that 2 of the smalllung metastases have disappeared while 1 of the larger lesions appearsto have diminished by about 40%. The metastases to the vertebrae arestill present, but the C4 and C5 lesions appear smaller by about 25%. Ofthe metastases to the ribs, 2 of 6 small lesions appear to be very muchdiminished in size, and are not certain as to being viable or smallareas of scar or necrosis. The patient's tumor markers, as well as herLDH titers, appear to show either stable or reduced levels, indicatingthat disease progression has been halted and there is also some evidenceof disease reduction. Subjectively, the patient is feeling much better,with less fatigue and bone pain and improved breathing. She hasexperienced some minimal nausea and vomiting after each therapy, whichresolved within a week. The only other side effect has been a transientthrombocytopenia, which also resolves within 7 days. She is beingobserved and will resume therapy cycles within 2 months.

Example 27 Treatment of Metastatic Colon Cancer with CombinationAnti-CEACAM5 and Anti-CEACAM6-CL2A-SN-38 Immunoconjugates

This patient has metastatic colonic cancer, with CT evidence of diseasein the liver (5 cm lesion in right lobe and 3 cm lesion in left lobe),as well as 2 metastases (2- and 3-cm sizes) to the right lung. Theprimary cancer of the colon was previously resected and the patient hadcourses of post-operative therapy because of metachronous metastases tothe liver and lungs. During therapy, the liver metastases grow and theone lung metastasis becomes two, so the patient is a candidate forexperimental chemoimmunotherapy. He is then begun on a course of doubleantibody-drug conjugates, labetuzumab (hMN-14)-CL2A-SN-38 andhMN-15-CL2A-SN-38, each given on alternate days at doses of 8 mg/kg,once weekly for 2 weeks, and then repeated monthly for 4 months. Twoweeks after therapy, the patient's status is evaluated by CT and labtests. The CT scans reveal that the large tumor in the right liver lobeis reduced by 50%, the tumor in the left lobe by about 33%, and the lungmetastases by about 20% cumulatively for both tumors. His blood CEAtiter is diminished from 22 ng/mL at onset of therapy to 6 ng/mL at thisfollowup. Subjectively, the patient states he is feeling stronger andalso appears to have more vigor in daily activities. Side effects aretransient thrombocytopenia and leucopenia, returning to normal rangeswithin 2 weeks after therapy, and several bouts of nausea and vomiting,controlled by anti-emetic medication. It is planned that the patientwill resume these therapy cycles in about 2 months, following anotherworkup for disease status.

Example 28 Continuous Infusion of Antibody-drug Conjugates

The patient was previously resected for a rectal carcinoma and receivespre- and post-operative radiochemotherapy as per conventional treatment.She has been free of tumor for four years, but now presents with 3 smallmetastatic lesions to the right liver lobe, discovered by routine CT andfollowup blood CEA values, which rise to 6.3 ng/mL from the 3.0 ng/mLpost initial therapy. She is given an indwelling catheter and acontinuous infusion of labetuzumab-CL2A-SN-28 at a dose of 2 mg/kg over17 days. She then receives a repeat continuous infusion therapy 5 weekslater, now for 3 weeks, at 1 mg/kg. Three weeks later, CT scans andblood CEA monitoring reveal that 1 of the liver metastases hasdisappeared and the other two are the same or slightly smaller. Theblood CEA titer now measures 2.4 ng/mL. She is not symptomatic, and onlyexperiences grade 2 nausea and vomiting while under therapy, and grade 2neutropenia, both resolving with time.

Example 29 Therapy of Advanced Metastatic Colon Cancer with Anti-CEACAM5Immunoconjugate

The patient is a 50-year-old male who fails prior therapies formetastatic colon cancer. The first line of therapy isFOLFIRINOX+AVASTIN® (built up in a stepwise manner) starting with IROX(Irinotecan+Oxaliplatin) in the first cycle. After initiating thistreatment the patient has a CT that shows decrease in the size of livermetastases. This is followed by surgery to remove tumor tissue. Adjuvantchemotherapy is a continuation of the first line regimen (without theIROX part) that resulted in a transient recurrence-free period. Afterabout a 1 year interval, a CT reveals the recurrence of livermetastases. This leads to the initiation of the second line regimen(FOLFIRI+Cetuximab). Another CT shows a response in liver metastases.Then RF ablation of liver metastases is performed, followed bycontinuation of adjuvant chemotherapy with FOLFIRINOX+Cetuximab,followed by maintenance Cetuximab for approximately one year. Another CTscan shows no evidence of disease. A further scan shows possible lungnodules, which is confirmed. This leads to a wedge resection of the lungnodules. Subsequently FOLFIRI+Cetuximab is restarted and continued.Later CT scans show both lung and liver metastases.

At the time of administration of the hMN-14-CL2A-SN-38 immunoconjugate,the patient has advanced metastatic colon cancer, with metastases ofboth lung and liver, which is unresponsive to irinotecan (camptothecin).The hMN-14-CL2A-SN-38 immunoconjugate is administered at a dosage of 12mg/kg, which is repeated every other week. The patient shows a partialresponse with reduction of metastatic tumors by RECIST criteria.

Of note is that only one patient in this 12 mg/kg (given every otherweek) cohort shows a grade 2 hematological (neutropenia) and mostpatients have grade 1 or 2 nausea, vomiting, or alopecia which are signsof activity of the antibody-drug conjugate, but well tolerated. Theeffect of the antibody moiety in improved targeting of the camptothecinaccounts for the efficacy of the SN-38 moiety in the cancer that hadbeen previously resistant to unconjugated irinotecan.

Example 30 Treatment of Metastatic Pancreatic Cancer withAnti-MUC5ac-CL2A-SN-38 Immunoconjugate

This 44-year-old patient has a history of metastatic pancreaticcarcinoma, with inoperable pancreas ductal adenocarcinoma in thepancreas head, and showing metastases to left and right lobes of theliver, the former measuring 3×4 cm and the latter measuring 2×3 cm. Thepatient is given a course of gemcitabine but shows no objectiveresponse. Four weeks later, he is given hPAM4-CL2A-SN-38 i.v. at a doseof 8 mg/kg twice-weekly for 2 weeks, with one week off, and thenrepeated for another 2 cycles. CT studies are done one week later andshow a total reduction in tumor mass (all sites) of 32% (partialresponse), alongside a drop in his blood CA19-9 titer from 220 atbaseline to 75 at the time of radiological evaluation. The patient showsonly grade 1 nausea and vomiting after each treatment with theantibody-drug conjugate, and a grade 2 neutropenia at the end of thelast treatment cycle, which resolves 4 weeks later. No premedication forpreventing infusion reactions is given.

Example 31 Use of hL243-CL2A-SN-38 to Treat Therapy-refractiveMetastatic Colonic Cancer (mCRC)

The patient is a 67-year-old man who presents with metastatic coloncancer. Following transverse colectomy shortly after diagnosis, thepatient then receives 4 cycles of FOLFOX chemotherapy in a neoadjuvantsetting prior to partial hepatectomy for removal of metastatic lesionsin the left lobe of the liver. This is followed by an adjuvant FOLFOXregimen for a total of 10 cycles of FOLFOX.

CT shows metastases to liver. His target lesion is a 3.0-cm tumor in theleft lobe of the liver. Non-target lesions included severalhypo-attenuated masses in the liver. Baseline CEA is 685 ng/mL.

After the patient signs the informed consent, hL243-CL2A-SN-38 (10mg/kg) is given every other week for 4 months. The patient experiencesnausea (Grade 2) and fatigue (Grade 2) following the first treatment andcontinues the treatment without major adverse events. The first responseassessment done (after 8 doses) shows shrinkage of the target lesion by26% by computed tomography (CT) and his CEA level decreases to 245ng/mL. In the second response assessment (after 12 doses), the targetlesion has shrunk by 35%. His overall health and clinical symptoms areconsiderably improved.

Example 32 Treatment of Relapsed Follicular Lymphoma withIMMU-114-CL2A-SN-38 (Anti-HLA-DR-SN-38)

After receiving R-CHOP chemotherapy for follicular lymphoma presentingwith extensive disease in various regional lymph nodes (cervical,axillary, mediastinal, inguinal, abdominal) and marrow involvement, this68-year-old man is given the experimental agent, IMMU-114-CL2A-SN-38(anti-HLA-DR-CL2A-SN-38) at a dose of 10 mg/kg weekly for 3 weeks, witha rest of 3 weeks, and then a second course for another 3 weeks. He isthen evaluated for change in index tumor lesions by CT, and shows a 23%reduction according CHESON criteria. The therapy is repeated for another2 courses, which then shows a 55% reduction of tumor by CT, which is apartial response.

Example 33 Treatment of Relapsed Chronic Lymphocytic Leukemia withIMMU-114-CL2A-SN-38

A 67-year-old man with a history of CLL, as defined by the InternationalWorkshop on Chronic Lymphocytic Leukemia and World Health Organizationclassifications, presents with relapsed disease after prior therapieswith fludarabine, dexamethasone, and rituximab, as well as a regimen ofCVP. He now has fever and night sweats associated with generalized lymphnode enlargement, a reduced hemoglobin and platelet production, as wellas a rapidly rising leukocyte count. His LDH is elevated and thebeta-2-microglobulin is almost twice normal. The patient is giventherapy with IMMU-114-CL2A-SN-38 conjugate at a dosing scheme of 8 mg/kgweekly for 4 weeks, a rest of 2 weeks, and then the cycle repeatedagain. Evaluation shows that the patient's hematological parameters areimproving and his circulating CLL cells appear to be decreasing innumber. The therapy is then resumed for another 3 cycles, after whichhis hematological and lab values indicate that he has a partialresponse.

Example 34 Use of hMN-15-CL2A-SN-38 to Treat Refractory, Metastatic,Non-small Cell Lung Cancer

The patient is a 58-year-old man diagnosed with non-small cell lungcancer. He is initially given chemotherapy regimens of carboplatin,bevacizumab for 6 months and shows a response, and then afterprogressing, receives further courses of chemotherapy with carboplatin,etoposide, TAXOTERE®, gemcitabine over the next 2 years, with occasionalresponses lasting no more than 2 months. The patient then presents witha left mediastinal mass measuring 5.5×3.5 cm and pleural effusion.

After signing informed consent, the patient is given hMN-15-CL2A-SN-38at a dose of 12 mg/kg every other week. During the first two injections,brief periods of neutropenia and diarrhea are experienced, but theseresolve or respond to symptomatic medications within 2 days. After atotal of 6 infusions of hMN-15-SN-38, CT evaluation of the target lesionshows a 22% reduction. The patient continues with this therapy foranother two months, when a partial response of 45% is noted by CT.

Example 35 Treatment of Follicular Lymphoma Patient with hA19-CL2A-SN-38

A 60-year-old male presents with abdominal pain and the presence of apalpable mass. The patient has CT and FDG-PET studies confirming thepresence of the mass with pathologic adenopathies in the mediastinum,axillary, and neck nodes. Lab tests are unremarkable except for elevatedLDH and beta-2-microglobulin. Bone marrow biopsy discloses severalparatrabecular and perivascular lymphoid aggregates. These arelymphocytic with expression of CD20, CD19, and CD10 by immunostaining.The final diagnosis is grade-2 follicular lymphoma, stage IVA, with aFLIPI score of 4. The longest diameter of the largest involved node is 7cm. The patient is given a humanized anti-CD19 monoclonal antibody IgG(hA19) conjugated with SN-38 (6 drug molecules per IgG) with a CL2Alinker. The dosing is 6 mg/kg weekly for 4 consecutive weeks, two weeksoff, and then repeated cycles of 4 treatment weeks for every 6 weeks.After 5 cycles, bone marrow and imaging (CT) evaluations show a partialresponse, where the measurable lesions decrease by about 60% and thebone marrow is much less infiltrated. Also, LDH and beta-2-microglobulintiters also decrease.

Example 36 Treatment of Relapsed Precursor B-cell ALL withhA19-CL2A-SN-38

This 51-year-old woman has been under therapy for precursor,Philadelphia chromosome-negative, B-cell ALL, which shows the ALL cellsstain for CD19, CD20, CD10, CD38, and CD45. More than 20% of the marrowand blood lymphoblasts express CD19 and CD20. The patient has receivedprior therapy with clofarabine and cytarabine, resulting in considerablehematological toxicity, but no response. A course of high-dosecytarabine (ara-C) was also started, but could not be tolerated by thepatient. She is given hA19-CL2A-SN-38 therapy at weekly doses byinfusion of 6 mg/kg for 5 weeks, and then a 2-week rest, with repetitionof this therapy two more times. Surprisingly, she shows improvement inher blood and marrow counts, sufficient for a partial response to bedetermined. After a rest of 2 months because of neutropenia (grade 3),therapy resumes at 8 mg/kg every other week for another 4 courses. Atthis time, she is much improved and is under consideration formaintenance therapy to try to bring her to a stage where she could be acandidate for stem-cell transplantation.

Example 37 Treatment of Lymphoma with Anti-CD22-CL2A-SN-38Immunoconjugate

The patient is a 62 year-old male with relapsed diffuse large B-celllymphoma (DLBCL). After 6 courses of R-CHOP chemoimmunotherapy, he nowpresents with extensive lymph node spread in the mediastinum, axillary,and inguinal lymph nodes. He is given epratuzumab-CL2A-SN-38 (anti-CD22)at a dose of 12 mg/kg weekly×3, with one week off, and then repeatedagain for another two cycles. One week later, the patient is evaluatedby CT imaging, and his total tumor bulk is measured and shows a decreaseof 35% (partial response), which appears to be maintained over the next3 months. Side effects are only thrombocytopenia and grade 1 nausea andvomiting after therapy, which resolve within 2 weeks. No pretherapy forreducing infusion reactions is given.

Example 38 Combination Therapy of Follicular Lymphoma with Veltuzumaband Epratuzumab-CL2A-SN-38 in the Frontline Setting

A 35-year-old woman is diagnosed with a low-grade and good FLIPI scorefollicular lymphoma, presenting in her cervical lymph nodes, bothaxilla, and mediastinum. Her spleen is not enlarged, and bone marrowbiopsy does not disclose disease involvement. She is symptomatically notmuch affected, with only periods of elevated temperature, night sweats,and somewhat more fatigued than usual. Her physician decides not toundertake a watch-and-wait process, but to give this woman aless-aggressive therapy combining a subcutaneous course of the humanizedanti-CD20 monoclonal antibody, veltuzumab, weekly×4 weeks (200 mg/m²)combined with two weekly courses of the anti-CD22epratuzumab-CL2A-SN-38, each infusion being a dose of 8 mg/kg. Thiscombination therapy is repeated 2 months later, and after this thepatient is evaluated by CT and FDG-PET imaging studies, as well as abone marrow biopsy. Surprisingly, about a 90% reduction of all diseaseis noted, and she then is given another course of this combinationtherapy after a rest of 4 weeks. Evaluation 4 weeks later shows aradiological (and bone marrow biopsy) complete response. Her physiciandecides to repeat this course of therapy 8 months later, andradiological/pathological tests show a sustained complete remission.

Example 39 Frontline Therapy of Follicular Lymphoma UsingVeltuzumab-CL2A-SN-38

The patient is a 41-year-old woman presenting with low-grade follicularlymphoma, with measurable bilateral cervical and axillary lymph nodes(2-3 cm each), mediastinal mass of 4 cm diameter, and an enlargedspleen. She is given 3 courses of veltuzumab-CL2A-SN-38(anti-CD20-CL2A-SN-38) therapy, with each course consisting of 10 mg/kginfused once every 3 weeks. After completion of therapy, her tumormeasurements by CT show a reduction of 80%. She is then given 2additional courses of therapy, and CT measurements indicate that acomplete response is achieved. This is confirmed by FDG-PET imaging.

Example 40 Therapy of Relapsed DLBCL with 1F5 Humanized AntibodyConjugated with CL2A-SN-38

A 53-year-old woman presents with recurrent diffuse large B-celllymphoma at mediastinal and abdominal para-aortic sites 8 months aftershowing a partial response to R-CHOP chemotherapy given for 6 cycles.She refuses to have more cytotoxic chemotherapy, so is given a mildertherapy consisting of 10 mg/kg humanized 1F5 anti-CD20 monoclonalantibody, conjugated to about 6 molecules of SN-28 per molecule ofantibody with CL2A linker, once weekly every other week for 5 infusions.CT and FDG-PET studies indicate a further reduction of her lymphomas by40%, so after a rest period of 4 weeks, therapy is resumed at a dose of8 mg/kg every 3 weeks for a total of 5 infusions. Evaluation of herdisease reveals a reduction of about 80%.

Example 41 Therapy of Relapsed Chronic Lymphocytic Leukemia withRituximab-CL2A-SN-38

A 62-year-old man with an 8-year history of CLL, having responded in thepast to fludarabine, cyclophosphamide, and rituximab therapy, and afterrelapse to ibrutinib for a partial response lasting 9 months, presentswith progressing disease. The patient is given rituximab-CL2A-SN-38monotherapy at a schedule of 12 mg/kg every 2 weeks for 3 courses,reduced to 8 mg/kg every other week for another 4 courses. Sustainedimprovement in cytopenias, reflected by more than 50% or a hemoglobinlevel higher than 11 g per deciliter, an absolute neutrophil counthigher than 1500 cells per cmm, or a platelet count higher than 11 k/cmmis observed, which was durable for about 9 months.

Example 42 Frontline Therapy of DLBCL with Veltuzumab-CL2A-SN-38Combined with Bendamustine

A 59-year-old man presents with multiple sites of DLBCL, includingchest, abdominal, inguinal lymph nodes, and enlarged spleen, asconfirmed by CT, FDG-PET, and immunohistological/pathological diagnoses.Bendamustine is given at a dose of 90 mg/m² on days 1 and 2, combinedwith veltuzumab-CL2A-SN-38 at a dose of 6 mg/kg on days 7 and 14, givenevery 4 weeks for four cycles. Evaluation radiologically thereaftershows a partial response. After a rest of 2 months, the therapy isrepeated for another 2 cycles, and radiological assessment then shows acomplete response. Cytopenias, mostly neutropenia, is manageable anddoes not achieve a grade 3 level.

Example 43 Frontline Therapy of Mantle Cell Lymphoma (MCL) withVeltuzumab-CL2A-SN-38 Combined with Lenalidomide

The patient is a 68-year-old man diagnosed with MCL after presentingwith a GI complaint and lethargy. Colonoscopy discloses a 7-cm cecalmass, and his workup reveals that he has Stage IV disease. He is given acombination therapy of lenalidomide, 25 mg orally daily on days 1 to 21every 28 days. After two cycles, he is given veltuzumab-CL2A-SN-38 everyother week at a dose of 10 mg/kg for 3 treatments, with a 2-week rest.This is then repeated again. Two weeks after completion of this therapy,the patient shows a partial response of his measured index lesion andreduction of other lymph nodes visualized. Four months later,lenalidomide therapy is repeated for 21 days, followed by 2 courses ofveltuzumab-SN-38. His disease is then shown to be reduced even further,although not yet a complete response.

Example 44 Epratuzumab-SN-38 Therapy of a Patient withRelapsed/Refractory Diffuse Large B-cell Lymphoma (DLBCL)

A 65-year-old man with symptoms of weight loss undergoes a biopsy of anepigastric mass, which is diagnosed as a diffuse large B-cell lymphoma.He is treated with 6 cycles of standard R-CHOP (rituximab,cyclophosphamide, doxorubicin, vincristine, and prednisone). He hasprolonged and persistent neutropenia (700 ANC) with mildthrombocytopenia (50-70 k/mL), but no real anemia. His IPI is high. Theepigastric mass does not show any change following this therapy, and heis put on a mild treatment regimen of anti-CD22 epratuzumab-CL2A-SN-38.This is a regimen of 4 mg/kg infused every other week for 4 infusions,then once every third week for another 3 infusions. His epigastriclymphoma is measured by CT one week later and shows a marked reductionby 52%. The patient is continues this treatment every third week foranother 3 months, and continues to show this reduction of his lymphomamass with stabilization of his weight and improvement of his energy andactivities.

Example 45 Humanized RFB4 Therapy of a Patient with Relapsed FollicularLymphoma

A 42-year-old woman presents with a sharp, constant and severe pain inher lower abdomen which radiates to her back. Laboratory tests areunremarkable, but an abdominal ultrasound shows a heterogeneous solidmass in the anterior lower left, measuring 7.5×6.2×7.0 cm. CT scansreveal a large mass within the left small-bowel mesentery, withinvolvement of adjacent lymph nodes. A CT-guided needle biopsy of themass shows that it is a follicular lymphoma, grade 3.Immunohistochemistry shows a B-cell type with positive results for CD19,CD20, CD22, Bcl-2 and Bcl-6. PET studies reveal no disease above thediaphragm, in the bone marrow, or in the spleen, but bone marrow biopsydoes confirm involvement of lymphoma. The patient undergoes 6 cycles ofR-CHOP chemotherapy, resulting in a complete response to this therapy 4months later. However, 10 months later, she undergoes a relapse, withrecurrence of her abdominal mass and adjacent lymph nodes, as well as anenlarged spleen and more bone marrow involvement as determined by PETand biopsy studies. She now begins a course of therapy with thehumanized anti-CD22 monoclonal antibody, RFB4, conjugated with 6 SN-38molecules per IgG using CL2A linker, at a dose of 8 mg/kg weekly for 3weeks, and then continued at 8 mg/kg every other week for another 4treatments. Two weeks later, she undergoes CT and FDG-PET studies, andher abdominal lesions and spleen show a reduction of 40%, and a generaldecrease of bone marrow involvement. After a rest of 4 weeks, therapy at4 mg/kg weekly for 4 weeks, followed by 6 mg/kg every other week foranother 5 treatments are implemented, with a further measurablereduction of the sum of the sizes of all measured lesions by a total of60%. The patient continues on a maintenance therapy of hRFB4-CL2A-SN-38of 8 mg/kg once monthly for the next 5 months, and maintains hertherapeutic response.

Example 46 Epratuzumab-CL2A-SN-38 Therapy of a Patient withRelapsed/Refractory Acute Lymphoblastic Leukemia

A 29-year-old male with CD22+ precursor B-cell acute lymphoblasticleukemia (ALL) has not responded to therapy with PEG-asparaginase,cyclophosphamide, daunorubicin, cytarabine (ara-C), vincristine,leucovorin, prednisone, methotrexate, and 6-mercaptopurine, andsupportive therapy with G-CSF (Neupogen), given as induction/maintenancetherapies under a modified Larson protocol. The patient's leukemia isPhiladelphia chromosome-negative. Based on blood and marrow leukemiablast counts, the patient shows only a minimal response, with diseaseprogressing 4 months later. He is then given weekly dosing ofepratuzumab-CL2A-SN-38 at an initial schedule of 6 mg/kg for 4 weeks,and then reduced to 6 mg/kg every-other-week for an additional 6infusions. The patient is then evaluated by blood and marrow leukemicblasts as well as FDG studies of spleen size and bone marrowinvolvement, and it appears that a partial response is achieved, withconcomitant improvement in the patient's general signs and symptoms.This therapy then continues over the next 8 months, but at 5 mg/kg everyother week, with 3 weeks on therapy and 2 weeks rest, and a completeremission is achieved. The patient is now being evaluated as a candidatefor hematopoietic stem cell transplantation.

Example 47 Humanized RFB4-CL2A-SN-38 Therapy of a Patient withRelapsed/Refractory Acute Lymphoblastic Leukemia

After failing to respond to HIDAC (high-dose ara-C therapy), this20-year-old man with precursor B-cell acute lymphoblastic leukemia isgiven humanized anti-CD22 therapy with hRFB4 IgG conjugated to SN-38(average of 6 drug molecules per IgG), at a dosing schedule of 10 mg/kgweekly for two weeks, then 1 week rest, followed by infusions of 10mg/kg every other week for an additional 5 treatments. The patient isthen evaluated for presence of blood and marrow leukemic blast cells,and shows a >90% reduction. After a rest of 4 weeks, this therapy courseis repeated, and the evaluation 4 weeks later shows a complete responsewith no minimal residual disease, as measured by PCR.

Example 48 Consolidation Therapy with Epratuzumab-CL2A-SN-38 in a DLBCLPatient Receiving R-CHOP Chemotherapy

This 56-year-old woman with bilateral cervical adenopathy and cervicallymph nodes measuring 1.5 to 2.0 cm, as well as a right axillary lymphnode of 3 cm, as well as retroperitoneal and bilateral pelvic lymphnodes measuring 2.5 to 3.0 cm, is diagnosed with stage 3 diffuse largeB-cell lymphoma that is positive for CD20 and CD22. She is put on astandard R-CHOP chemotherapy regimen given every 21 days with filgrastimand prophylactic antibiotics. After receiving 6 cycles of this therapy,the patient is given a rest period of 2 months, and then is put onconsolidation therapy with 8 mg/kg epratuzumab-CL2A-SN-38, infused everyother week for 3 treatments. Whereas the response after the R-CHOPchemotherapy is minimal (less than 30% change in measured lesions),consolidation therapy with epratuzumab-CL2A-SN-38 results in a partialresponse (>50% decrease in sum of all index lesions). After a rest of 3months, this course of therapy with epratuzumab-CL2A-SN-38 is repeated,with the patient again given filgrastim and prophylactic antibiotics,and maintains her good remission.

Example 49 Treatment of Relapsed Metastatic Testicular Cancer withIMMU-31-CL2A-SN-38

The patient is a 30-year-old man with a history of resected testicularcancer of his right testicle, with synchronous metastases to both lungsthat respond well to combination chemotherapy. At diagnosis, his bloodtiter of alpha-fetoprotein (AFP) is elevated at 1,110 ng/mL, butdecreases to 109 ng/mL after successful therapy. He now presents with agradually rising AFP titer over a period of 3 years, so CT and FDG-PETscans of his body are made, revealing recurrence of lung metastases toboth lungs. He receives therapy with the anti-AFP antibody, IMMU-31 IgG,conjugated with SN-38 at 6 drug molecules per IgG. He receives weeklydoses of 12 mg/kg of this antibody-drug conjugate for 3 weeks of a4-week cycle, repeated for another cycle but with a reduction of thetherapeutic to 10 mg/kg. This is then repeated for another 2 cycles. Twoweeks later, radiological examination of his lungs reveals that themetastases have disappeared. His blood AFP titer is now 18 ng/mL. Thepatient returns to normal activity with a complete response having beenachieved.

Example 50 Treatment of Relapsed Metastatic Hepatocellular Carcinomawith IMMU-31-CL2A-SN-38

A 58-year-old male with a history of hepatitis B infection, alcoholexcess and smoking, leads first to liver cirrhosis and then a diagnosisof hepatocellular carcinoma. At the time he presents after having aportion of his liver resected, there are also regional lymph nodesinvolved. The patient receives a course of sorafenib therapy, indicatessome general improvement, but does not have any reduction of hisregional lymph node or 2 lung (right lung) metastases. CT of the liveralso suggests that there may be a recurrence in the remaining liverparenchyma. This patient is now given 3 courses of therapy withIMMU-31-CL2A-SN-38, each comprising a schedule of weekly 16 mg/kg for 2weeks of a 4-week cycle. After the 3 courses comprising 6 doses, thepatient is reevaluated and shows a decrease in his circulating AFP titerfrom the baseline value of 2,000 ng/mL to 170 ng/mL, as well as a 20%reduction of the sum of his measured index lesions. After a rest of 2months, another course of therapy of 3 cycles, but with a reduction ofthe dose to 1 mg/kg per infusion, is instituted. One month later, thereis a greater reduction of all measured lesion, to 35% of baseline, aswell as a slight decrease in the AFP blood titer to 100 ng/mL. Thepatient is going on maintenance therapy of one dose per month for aslong as there is no disease progression or limiting toxicities.

Example 51 Immunoconjugate Storage

The conjugates described in Example 2 were purified and buffer-exchangedwith 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, and furtherformulated with trehalose (25 mM final concentration) and polysorbate 80(0.01% v/v final concentration), with the final buffer concentrationbecoming 22.25 mM as a result of excipient addition. The formulatedconjugates were lyophilized and stored in sealed vials, with storage at2° C. 8° C. The lyophilized immunoconjugates were stable under thestorage conditions and maintained their physiological activities.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated byreference.

What is claimed is:
 1. A method to produce a compound, CL2A-SN-38, ofthe structure,

comprising performing a reaction scheme as shown:

wherein the reaction scheme is performed in the absence oftriphenylphosphine; further comprising: (i) using a 1.1-fold molarexcess of tetrabutylammonium fluoride to remove a silyl protecting groupand convert intermediate 6 to intermediate 7; and (ii) performing threewashes of an organic extract comprising intermediate 6 with 0.05 Msodium acetate buffer, pH 5.3.
 2. The method of claim 1, furthercomprising performing two washes of an organic extract comprisingintermediate 7 with 0.25 M sodium citrate buffer, pH
 6. 3. The method ofclaim 1, wherein organic extracts comprising intermediate 7 orintermediate 8 are not washed with water.
 4. The method of claim 1,further comprising purifying intermediate 7 by step elution from asilica gel column with a mixture of dichloromethane, ethyl acetate andmethanol.
 5. The method of claim 1, further comprising forming abiphasic mixture comprising: a) copper sulfate and sodium ascorbate inwater; and b) intermediate 7, intermediate 8 and 2,6-collidine indichloromethane.
 6. The method of claim 5, further comprising stirringthe biphasic mixture overnight at room temperature.
 7. The method ofclaim 1, further comprising purifying intermediate 9 by EDTA washing andchromatography.
 8. The method of claim 1, further comprisingconcentrating intermediate 9 to a solid for storage, prior to removing asilyl group from intermediate 9 to form CL2A-SN-38.
 9. The method ofclaim 8, further comprising (i) forming a solution of solid intermediate9; and (ii) reacting intermediate 9 in solution with dichloroacetic acidand anisole to form CL2A-SN-38.
 10. The method of claim 9, furthercomprising precipitating the CL2A-SN-38 by dropwise addition oftent-butyl methyl ether (t-BME).
 11. The method of claim 1, furthercomprising reacting a maleimide moiety of CL2A-SN38 with a protein orpeptide to make an SN38-conjugated protein or peptide.
 12. The method ofclaim 11, wherein the maleimide moiety reacts with a reduced sulfhydrylon the protein or peptide.
 13. The method of claim 11, wherein theprotein or peptide is an antibody or an antigen-binding antibodyfragment and the SN-38 conjugated antibody or antibody fragment is animmunoconjugate.
 14. The method of claim 13, further comprisingpurifying the immunoconjugate by tangential flow filtration (TFF). 15.The method of claim 14, wherein the TFF is performed with a 50,000dalton molecular weight cut-off membrane using 25- to 30-diafiltrationvolumes of buffer.
 16. The method of claim 13, further comprisingformulating the immunoconjugate in Good's biological buffer at a pH of6.0 to 7.0, and lyophilizing the immunoconjugate for storage.
 17. Themethod of claim 16, wherein the Good's biological buffer is selectedfrom the group consisting of 2-(N-morpholino)ethanesulfonic acid (MES),3-(N-morpholino) propanesulfonic acid (MOPS),4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), and1,4-piperazinediethanesulfonic acid (PIPES), in the pH range of 6-7,preferably in the pH range of 6.5 to 7, and at a buffer concentration of10-100 mM, preferably 25 mM.
 18. The method of claim 17, wherein thebuffer is 25 mM MES buffer, pH 6.5.
 19. The method of claim 13, whereinthe antibody is a bispecific antibody or a monoclonal antibody.
 20. Themethod of claim 13, wherein the antibody fragment is selected from thegroup consisting of F(ab')₂, F(ab)₂, Fab', Fab, Fv, scFv, single domainantibody and half-molecule of IgG4 antibody.
 21. The method of claim 13,wherein the antibody or antibody fragment is attached to between 1 and12 copies of CL2A-SN38.
 22. The method of claim 13, wherein the antibodyor antibody fragment is attached to between 6 and 8 copies of CL2A-SN38.23. The method of claim 13, wherein the antibody or antibody fragment isattached to between 1 and 5 copies of CL2A-SN38.
 24. The method of claim13, wherein the antibody is an anti-cancer antibody, an anti-infectiousdisease antibody, or an anti-autoimmune disease antibody.
 25. The methodof claim 13, wherein the antibody is selected from the group consistingof LL1 (anti-CD74), LL2 (anti-CD22), RFB4 (anti-CD22), RS7 (anti-EGP-1),PAM4 (anti-MUC5AC), KC4 (anti-mucin), A19 (anti-CD19), A20 (anti-CD20),MN-14 (anti-CEACAM5), MN-15 (anti-CEACAM6), MN-3 (anti-CEACAM6), R1(anti-IGF-1R), Mu-9 (anti-CSAp), Immu 31 (anti-AFP), CC49 (anti-TAG-72),J591 (anti-PSMA), HuJ591 (anti-PSMA), AB-PG1-XG1-026 (anti-PSMA dimer),D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX) and hL243(anti-HLA-DR).
 26. The method of claim 13, wherein the antibody orantibody fragment binds to an antigen selected from the group consistingof carbonic anhydrase IX, alpha-fetoprotein (AFP), α-actinin-4, ART-4,B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCL19,CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16,CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37,CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64,CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132,CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4,CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp), CEACAM5,CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M,Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor,G250 antigen, GAGE, gp100, GRO-β, H2B, H3, H4, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG), HER2/neu, HMGB-1, hypoxia inducible factor(HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R,IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15,IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KS1-4,Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE,MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B,MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3,NCA66, NCA95, NCA90, PD-1, PD-L1, PD-1 receptor, placental growthfactor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF,ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin,survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen,Thomson-Friedenreich antigen, tumor necrosis antigens, VEGFR, ED-Bfibronectin, WT-1 , 17-1A-antigen, complement factors C3, C3a, C3b, C5a,C5, bcl-2, bcl-6, Kras, and an oncogene product.